Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Extended Experimental ProceduresReagentsFor western blotting the following antibodies were used: rabbit anti-ppMEK1/2 and mouse anti-myc 9B11 (Cell Signaling Technology); mouse anti-NRAS (C-20), rabbit anti-ERK2 (C-14), rabbit anti-ARAF (C-20), mouse anti-BRAF (F-7) (Santa Cruz Biotechnology); mouse anti-Tubulin, and mouse anti-ppERK1/2 (Sigma); mouse anti-CRAF (for western blotting) (BD Transduction Laboratories).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For immunoprecipitation, the following antibodies were used: rabbit anti-myc (Abcam); rabbit anti-CRAF (C-20; Santa Cruz Biotechnology); mouse anti-BRAF (F-7) (Santa Cruz Biotechnology).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Calf intestinal phosphatase (CIP) was from New England Biolabs (NEB).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	PD184352, sorafenib and PLX4720 were synthesized in-house; 885-A was synthesized by Evotec AG (Abingdon, UK).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	All drugs were prepared in DMSO.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Synthetic routes are available on request.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Expression ConstructsThe expression vectors for wild-type human CRAF and wild-type human BRAF, pEFm/CRAF and pEFm/BRAF respectively have been described (Marais et al., 1995).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Briefly, the vector backbone is pUC19 and the elongation factor 1α (EF1α) promoter is used to drive exogenous protein expression.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The vector includes the first intron from human EF1α to assist mRNA processing during expression.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The β-globin 5′ and 3′ untranslated regions (UTRs) are used to provide a strong translation start site (5′ UTR), and also to provide mRNA stability and a poly adenylation signal (3′ UTR).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The vector introduces an amino-terminal myc-epitope tag (EQKLISEEDL) onto the exogenously expressed protein.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The BRAF coding region includes the alternatively spliced exons 1 and 2 but not exons 8b or 10a and various modifications were introduced to provide additional restriction sites (without changing the amino acid sequence) and alterations to the 3′-UTR to allow easier manipulation of this construct.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Standard PCR-directed mutagenesis approaches were used to generate the various mutations used in the study and all mutations were verified by automated dideoxy sequencing.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The expression vector pMCEF/FLAG/CRAF uses the same expression cassette, but the backbone also possesses a neo resistance cassette to facilitate selection in the presence of G418.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	In addition, a version of this vector was used that incorporates a FLAG (DYKDDDKGS), rather than a myc-epitope tag.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Cell CultureHuman cell lines were cultured in DMEM (A375, WM852, HCT116, SW620, PMWK, SKMel24, SKMel28, D25) or RPMI (D04, MM485, MM415, WM1791c) supplemented with 10% fetal bovine serum.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	African green monkey kidney COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	All cell lines were incubated at 37°C and 10% CO2.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For inhibitor treatment, the drugs were dissolved in DMSO and added to the medium for 2-5 hr.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	When two compounds were used, the first was added 30 min prior to the second.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For cell growth assays, cells were seeded in 96-well plates and treated with drug in quadruplicate in a 10-point titration assay for 5 days.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The amount of growth (% DMSO controls) was determined using sulphorhodamine B reagent (Monks et al., 1991) as follows.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	1,000-10,000 cells (depending on cell type) were plated into 96-well plates in 100 μL medium.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	After 24 hr, compounds prepared in DMSO (10mM stocks) were serially diluted in culture medium at 2× the final required concentrations and 100μL were added to the cells to nine final concentrations of 0.005 μM-100 μM. After a further 5 days, the cells are fixed in trichloroacteic acid (10%), and stained with sulforhodamine-B (0.1%).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	After rinsing, the bound stain was disolved using 100 μL 10 mM Tris (pH 8.0) and the absorbance at 540 nm determined.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The data were analyzed by nonlinear regression to a four-parameter logistic equation (Graphpad Prism, Graphpad Software Inc., San Diego, CA, USA) and the GI50 value determined.siRNA Transfections3 × 10 D04 cells per 35 mm diameter well were seeded in 2ml growth medium the day before transfection.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The cells were either mock-transfected or transfected with 6nM CRAF-specific (5′-AAGCACGCTTAGATTGGAATA-3′) or NRAS-specific (5′-CATGGCACTGTACTCTTCTCG-3′) siRNA using INTERFERin as recommended by the manufacturer (Polyplus Transfection SA).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Briefly, 0.6 μl of 20 μM siRNA and 6 μl of INTERFERin were combined in a total of 200 μl serum free medium in RNase-free tubes.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The mix was vortexed for 10 s and incubated for 5-10 min before adding the complexes dropwise to the cells.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The cells were serum-starved the day after transfecting and extracts were prepared 48 hr after transfection.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	DNA TransfectionsFor transient protein expression in D04 cells, Lonza Nucleofector Technology (Lonza, Cologne AG) was used.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	2 μg of DNA was mixed with 1x10 cells resuspended in 100 μl of Nucleofection Solution V in an Amaxa-certified cuvette and transfected using program T030.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The cells were re-plated into 35mm diameter tissue culture wells and incubated for 48 hr before preparation of cell extracts.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For generation of stable lines, D04 cells were transfected using Effectene (Invitrogen) and selected in G418.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	3-4x10 cells were plated in 35 mm diameter wells and incubated overnight.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	0.4 μg of DNA diluted into 100 μl of DNA condensation buffer (EC) and 3.2 μl enhancer were mixed vigorously and incubated for 2-5 min.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	10 μl Effectene reagent was added and the mixture was incubated for another 5-10 min.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The cells were washed with 2ml PBS and 1.6ml fresh serum containing medium was added.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The DNA complexes were diluted with 600 μl of culture medium and the mixture added to the cells drop-wise.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	After six hours, the medium was replaced with 2ml of fresh growth medium.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	After 48 hr, the cells were replated into several 10cm dishes in a 10-fold dilution series and incubated in G418 (1mg/ml) for selection.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The medium was refreshed weekly and after 2-3 weeks, single colonies were selected and expanded.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For transient expression in COS-7 cells, Lipofectamine (Invitrogen) was used.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	2x10 cells were plated into 35mm diameter wells and incubated overnight.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	75 to 200 ng of expression plasmid (depending on construct) was mixed with empty vector to a total of 700 ng DNA in 16μl PBS.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Typically, 3 μl of Lipofectamine in 13 μl of PBS was added to the DNA on the surface of a bacterial plate and incubated (Lipofectamine is inactivated by binding to polypropylene) for 15 min at room temperature.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The cells were washed twice with 1ml serum-free DMEM, and then overlaid with 800 μl of serum free DMEM.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	200 μl of serum free DMEM was added to the DNA:Lipofectamine mix, and the total volume was added to the cells.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	After six hours, the complexes were removed and replaced with 2ml of normal culture medium.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Cell extracts were prepared two days following transfection.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Preparation of Cell LysatesCulture medium was aspirated from cells and cells were placed on ice and washed three times in ice-cold PBS.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Depending on the assays, the cells were scraped into 50-200 μl Nonidet P40 (NP40) extraction buffer (Table S3) and incubated on ice for five minutes.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The cells were sheared by passing through a pipette tip several times and the samples were centrifuged at 20,000 × g for 5 min at 4°C and the soluble fraction was harvested.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	RAF CoimmunoprecipitationsImmunoprecipitations were performed in 300 μl cell lysates from one 35mm diameter well for endogenous protein or from 2-3 wells for transfected protein.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Endogenous BRAF or CRAF were immunoprecipitated with 2μg BRAF F-7 or 5μg CRAF C-20 respectively and myc-tagged BRAF and CRAF with 2 μg rabbit anti-myc antibody.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The antibody-protein complex was captured using 20 μl of a 1:1 Protein G sepharose 4B beads (Sigma-Aldrich) mixture in NP40 lysis buffer (Table S3) and immunoprecipitates (IPs) were mixed for 2 hr at 4°C on a rotation wheel.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Thereafter, the IPs were washed three times with 300 μl of NP40 lysis buffer (Table S3) before analysis on standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Specific bands were detected using fluorescent-labeled secondary antibodies (Invitrogen; Li-COR Biosciences) and analyzed using an Odyssey Infrared Scanner (Li-COR Biosciences).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For CIP treatment, immunoprecipitates were washed twice with NP40 lysis buffer (Table S3) and once in CIP buffer (Table S3).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Thereafter immunoprecipitates were incubated with 30 μl CIP buffer containing 5 units of CIP in the presence or absence of 0.2 mM Na3VO4 phosphatase inhibitor and 7mM EDTA.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Controls were incubated in CIP buffer without CIP.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The reactions were performed at 30°C for 30 min before analysis on SDS-PAGE.Cell Fractionation ExperimentsD04 cells were plated on two 10cm dishes per treatment and grown to confluency.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	After treatment, cells were washed three times with cold PBS, washed once with 20mM HEPES pH 7.4 and then lysed by scraping in 20mM HEPES pH 7.4 supplemented with protease inhibitors (1ml per 2 plates).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Cells were disrupted by passing them through a 9G syringe ten times, followed by another ten times through a 19G syringe (Terumo Medical).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Lysates were centrifuged at 900 × g for five minutes to pellet the nuclear proteins.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The supernatant was transferred to fresh 1.5ml ultracentrifuge tubes (Beckman Coulter) and 200 μl removed as a total lysate control.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The remainder was centrifuged at 100,000 × g for 30 min at 4°C to separate the cytosolic fraction from the membrane fraction.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The supernatant containing the cytosolic fraction was transferred to a fresh 1.5ml tube and the pelleted membrane fraction washed once in 20 mM HEPES pH 7.4 before resuspension in 200 μl 20mM HEPES pH 7.4/1% Triton X-100.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For analysis on SDS-PAGE, the concentration of protein was determined by Bradford protein assay (Bio-Rad Laboratories) using purified bovine serum albumin (BSA) as a standard as described by the manufacturer.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Equal amounts of protein were loaded for the cytosolic fraction and total cell lysate.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Three times as much protein was loaded for the membrane fraction.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	RAF Kinase AssaysThe in vitro kinase activity of endogenous RAF proteins or myc-tagged RAF proteins transiently expressed in COS-7 cells was measured using a coupled kinase cascade assay with GST-MEK, GST-ERK and myelin basic protein (MBP) (Sigma-Aldrich) as sequential substrates.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	ERK activation was quantified by measuring the incorporation of [P]-orthophosphate (PerkinElmer) into MBP.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For measurement of endogenous BRAF kinase activity, D04 or A375 cells were seeded in 10cm dishes and harvested in 300 μl of NP40 buffer (Table S3) as described above.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Protein concentrations were determined and equal amounts of protein were immunoprecipitated as described above.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For measurement of mutant BRAF kinase activities, COS-7 cells were transiently transfected with myc-tagged BRAF and cells in one 35 mm diameter well were harvested in 200μl of NP40 buffer (Table S3).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The relative concentrations of exogenously expressed RAF in these cell lysates were determined by quantitative western blotting using the myc antibody (Cell Signaling Technology) specified above.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Bands were quantified using the Odyssey infrared imaging system (LI-COR Biosciences).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Equivalent amounts of RAF were immunoprecipitated using rabbit myc antibody (Abcam) as specified above.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Endogenous and transiently transfected RAF proteins were immunoprecipitated in a total of 300 μl NP40 buffer (Table S3) for 2 hr at 4°C and immunoprecipitates were washed sequentially three times with chilled wash buffer (Table S3) containing decreasing concentrations of KCl (1M KCl, 0.1M KCl and no KCl).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The first-step reaction was initiated by addition of 20 μl MKK buffer (containing GST-MEK and GST-ERK, Table S3) to the beads and incubated at 30°C for 10 min in the case of myc-tagged BRAF or 30 min for endogenous BRAF and CRAF.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Reactions were terminated by the addition of 20 μl KILL buffer (Table S3), which contains EDTA to chelate Mg ions and inhibit kinase activity.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The reaction supernatants were collected from the beads and transferred into fresh tubes for the second step reaction.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	5 μl of supernatant was incubated with 25 μl MBP buffer containing [γ-P]ATP (PerkinElmer) for ten minutes at 30°C in triplicate to measure ERK activity.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The second reaction was terminated by spotting 20 μl of reaction mix onto a 1cm piece of P81 paper (VWR International), which was then dropped into 400ml 25mM orthophosphate solution.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The papers were washed three times in 400 ml 25 mM orthophosphate solution to remove the unincorporated ATP and the [P]-orthophosphate incorporated into MBP was determined using Cerenkov counting.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For transfected samples, the background counts were determined using lysates of cells transfected with the empty vector.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For endogenous protein, samples in which no RAF was immunoprecipitated were used.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Background values were removed and to ensure linearity, assays were used at below 50% saturation.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	To determine BRAF and BRAF sensitivity to 885-A, immunoprecipitated BRAF was preincubated with drug in KCl-free buffer for 10 min at room temperature prior to the first-step reaction.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	To measure the activity of purified BRAF, a 96-well DELFIA-based assay system was used.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Full-length rabbit MEK1 protein was expressed with a GST tag at the N-terminus and a C-terminal histidine tag in Escherichia coli JM109 bacteria and purified by nickel-agarose affinity chromatography.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Full length BRAF protein was generated by infection of SF9 insect cells with a recombinant baculovirus expressing full-length human BRAF with an N-terminal histidine tag and purified as above.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For the kinase assays, all incubations were at room temperature with shaking.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	4 μg GST-MEK1, 100-200ng purified BRAF and 1 μl inhibitor at the required concentrations (0.001 to 100 μM final concentration) were added to the wells of glutathione-coated plates and preincubated for 10 min.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	ATP in DELFIA assay buffer (20 μL; Table S3), to give a final concentration of 100 μM, was added to each well, and the plates were incubated for 45 min.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The plates were washed 3X with 200 μl 0.1% tween20/water.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Primary antibody (rabbit anti-phospho MEK1/2 diluted 1/2000, Cell Signaling Technologies) and Eu-labeled anti-rabbit secondary antibody (diluted 1/1000, Perkin-Elmer) were preincubated for 30 min and 100 μl was added to the plates and incubated for a further hour.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The plates were washed as before, and 100 μl DELFIA enhancement solution (Perkin-Elmer, Turku, Finland) was added.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	The plates were sealed and incubated for 30 min and europium counts measured on Spectramax M5 plate reader (Molecular Devices, Wokingham, UK).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Extended Experimental ProceduresReagentsFor western blotting the following antibodies were used: rabbit anti-ppMEK1/2 and mouse anti-myc 9B11 (Cell Signaling Technology); mouse anti-NRAS (C-20), rabbit anti-ERK2 (C-14), rabbit anti-ARAF (C-20), mouse anti-BRAF (F-7) (Santa Cruz Biotechnology); mouse anti-Tubulin, and mouse anti-ppERK1/2 (Sigma); mouse anti-CRAF (for western blotting) (BD Transduction Laboratories).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For immunoprecipitation, the following antibodies were used: rabbit anti-myc (Abcam); rabbit anti-CRAF (C-20; Santa Cruz Biotechnology); mouse anti-BRAF (F-7) (Santa Cruz Biotechnology).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Calf intestinal phosphatase (CIP) was from New England Biolabs (NEB).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

PD184352, sorafenib and PLX4720 were synthesized in-house; 885-A was synthesized by Evotec AG (Abingdon, UK).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

All drugs were prepared in DMSO.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Synthetic routes are available on request.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Expression ConstructsThe expression vectors for wild-type human CRAF and wild-type human BRAF, pEFm/CRAF and pEFm/BRAF respectively have been described (Marais et al., 1995).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Briefly, the vector backbone is pUC19 and the elongation factor 1α (EF1α) promoter is used to drive exogenous protein expression.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The vector includes the first intron from human EF1α to assist mRNA processing during expression.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The β-globin 5′ and 3′ untranslated regions (UTRs) are used to provide a strong translation start site (5′ UTR), and also to provide mRNA stability and a poly adenylation signal (3′ UTR).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The vector introduces an amino-terminal myc-epitope tag (EQKLISEEDL) onto the exogenously expressed protein.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The BRAF coding region includes the alternatively spliced exons 1 and 2 but not exons 8b or 10a and various modifications were introduced to provide additional restriction sites (without changing the amino acid sequence) and alterations to the 3′-UTR to allow easier manipulation of this construct.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Standard PCR-directed mutagenesis approaches were used to generate the various mutations used in the study and all mutations were verified by automated dideoxy sequencing.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The expression vector pMCEF/FLAG/CRAF uses the same expression cassette, but the backbone also possesses a neo resistance cassette to facilitate selection in the presence of G418.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

In addition, a version of this vector was used that incorporates a FLAG (DYKDDDKGS), rather than a myc-epitope tag.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Cell CultureHuman cell lines were cultured in DMEM (A375, WM852, HCT116, SW620, PMWK, SKMel24, SKMel28, D25) or RPMI (D04, MM485, MM415, WM1791c) supplemented with 10% fetal bovine serum.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

African green monkey kidney COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

All cell lines were incubated at 37°C and 10% CO2.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For inhibitor treatment, the drugs were dissolved in DMSO and added to the medium for 2-5 hr.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

When two compounds were used, the first was added 30 min prior to the second.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For cell growth assays, cells were seeded in 96-well plates and treated with drug in quadruplicate in a 10-point titration assay for 5 days.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The amount of growth (% DMSO controls) was determined using sulphorhodamine B reagent (Monks et al., 1991) as follows.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

1,000-10,000 cells (depending on cell type) were plated into 96-well plates in 100 μL medium.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

After 24 hr, compounds prepared in DMSO (10mM stocks) were serially diluted in culture medium at 2× the final required concentrations and 100μL were added to the cells to nine final concentrations of 0.005 μM-100 μM. After a further 5 days, the cells are fixed in trichloroacteic acid (10%), and stained with sulforhodamine-B (0.1%).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

After rinsing, the bound stain was disolved using 100 μL 10 mM Tris (pH 8.0) and the absorbance at 540 nm determined.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The data were analyzed by nonlinear regression to a four-parameter logistic equation (Graphpad Prism, Graphpad Software Inc., San Diego, CA, USA) and the GI50 value determined.siRNA Transfections3 × 10 D04 cells per 35 mm diameter well were seeded in 2ml growth medium the day before transfection.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The cells were either mock-transfected or transfected with 6nM CRAF-specific (5′-AAGCACGCTTAGATTGGAATA-3′) or NRAS-specific (5′-CATGGCACTGTACTCTTCTCG-3′) siRNA using INTERFERin as recommended by the manufacturer (Polyplus Transfection SA).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Briefly, 0.6 μl of 20 μM siRNA and 6 μl of INTERFERin were combined in a total of 200 μl serum free medium in RNase-free tubes.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The mix was vortexed for 10 s and incubated for 5-10 min before adding the complexes dropwise to the cells.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The cells were serum-starved the day after transfecting and extracts were prepared 48 hr after transfection.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

DNA TransfectionsFor transient protein expression in D04 cells, Lonza Nucleofector Technology (Lonza, Cologne AG) was used.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

2 μg of DNA was mixed with 1x10 cells resuspended in 100 μl of Nucleofection Solution V in an Amaxa-certified cuvette and transfected using program T030.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The cells were re-plated into 35mm diameter tissue culture wells and incubated for 48 hr before preparation of cell extracts.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For generation of stable lines, D04 cells were transfected using Effectene (Invitrogen) and selected in G418.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

3-4x10 cells were plated in 35 mm diameter wells and incubated overnight.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

0.4 μg of DNA diluted into 100 μl of DNA condensation buffer (EC) and 3.2 μl enhancer were mixed vigorously and incubated for 2-5 min.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

10 μl Effectene reagent was added and the mixture was incubated for another 5-10 min.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The cells were washed with 2ml PBS and 1.6ml fresh serum containing medium was added.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The DNA complexes were diluted with 600 μl of culture medium and the mixture added to the cells drop-wise.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

After six hours, the medium was replaced with 2ml of fresh growth medium.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

After 48 hr, the cells were replated into several 10cm dishes in a 10-fold dilution series and incubated in G418 (1mg/ml) for selection.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The medium was refreshed weekly and after 2-3 weeks, single colonies were selected and expanded.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For transient expression in COS-7 cells, Lipofectamine (Invitrogen) was used.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

2x10 cells were plated into 35mm diameter wells and incubated overnight.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

75 to 200 ng of expression plasmid (depending on construct) was mixed with empty vector to a total of 700 ng DNA in 16μl PBS.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Typically, 3 μl of Lipofectamine in 13 μl of PBS was added to the DNA on the surface of a bacterial plate and incubated (Lipofectamine is inactivated by binding to polypropylene) for 15 min at room temperature.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The cells were washed twice with 1ml serum-free DMEM, and then overlaid with 800 μl of serum free DMEM.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

200 μl of serum free DMEM was added to the DNA:Lipofectamine mix, and the total volume was added to the cells.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

After six hours, the complexes were removed and replaced with 2ml of normal culture medium.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Cell extracts were prepared two days following transfection.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Preparation of Cell LysatesCulture medium was aspirated from cells and cells were placed on ice and washed three times in ice-cold PBS.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Depending on the assays, the cells were scraped into 50-200 μl Nonidet P40 (NP40) extraction buffer (Table S3) and incubated on ice for five minutes.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The cells were sheared by passing through a pipette tip several times and the samples were centrifuged at 20,000 × g for 5 min at 4°C and the soluble fraction was harvested.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

RAF CoimmunoprecipitationsImmunoprecipitations were performed in 300 μl cell lysates from one 35mm diameter well for endogenous protein or from 2-3 wells for transfected protein.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Endogenous BRAF or CRAF were immunoprecipitated with 2μg BRAF F-7 or 5μg CRAF C-20 respectively and myc-tagged BRAF and CRAF with 2 μg rabbit anti-myc antibody.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The antibody-protein complex was captured using 20 μl of a 1:1 Protein G sepharose 4B beads (Sigma-Aldrich) mixture in NP40 lysis buffer (Table S3) and immunoprecipitates (IPs) were mixed for 2 hr at 4°C on a rotation wheel.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Thereafter, the IPs were washed three times with 300 μl of NP40 lysis buffer (Table S3) before analysis on standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Specific bands were detected using fluorescent-labeled secondary antibodies (Invitrogen; Li-COR Biosciences) and analyzed using an Odyssey Infrared Scanner (Li-COR Biosciences).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For CIP treatment, immunoprecipitates were washed twice with NP40 lysis buffer (Table S3) and once in CIP buffer (Table S3).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Thereafter immunoprecipitates were incubated with 30 μl CIP buffer containing 5 units of CIP in the presence or absence of 0.2 mM Na3VO4 phosphatase inhibitor and 7mM EDTA.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Controls were incubated in CIP buffer without CIP.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The reactions were performed at 30°C for 30 min before analysis on SDS-PAGE.Cell Fractionation ExperimentsD04 cells were plated on two 10cm dishes per treatment and grown to confluency.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

After treatment, cells were washed three times with cold PBS, washed once with 20mM HEPES pH 7.4 and then lysed by scraping in 20mM HEPES pH 7.4 supplemented with protease inhibitors (1ml per 2 plates).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Cells were disrupted by passing them through a 9G syringe ten times, followed by another ten times through a 19G syringe (Terumo Medical).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Lysates were centrifuged at 900 × g for five minutes to pellet the nuclear proteins.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The supernatant was transferred to fresh 1.5ml ultracentrifuge tubes (Beckman Coulter) and 200 μl removed as a total lysate control.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The remainder was centrifuged at 100,000 × g for 30 min at 4°C to separate the cytosolic fraction from the membrane fraction.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The supernatant containing the cytosolic fraction was transferred to a fresh 1.5ml tube and the pelleted membrane fraction washed once in 20 mM HEPES pH 7.4 before resuspension in 200 μl 20mM HEPES pH 7.4/1% Triton X-100.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For analysis on SDS-PAGE, the concentration of protein was determined by Bradford protein assay (Bio-Rad Laboratories) using purified bovine serum albumin (BSA) as a standard as described by the manufacturer.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Equal amounts of protein were loaded for the cytosolic fraction and total cell lysate.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Three times as much protein was loaded for the membrane fraction.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

RAF Kinase AssaysThe in vitro kinase activity of endogenous RAF proteins or myc-tagged RAF proteins transiently expressed in COS-7 cells was measured using a coupled kinase cascade assay with GST-MEK, GST-ERK and myelin basic protein (MBP) (Sigma-Aldrich) as sequential substrates.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

ERK activation was quantified by measuring the incorporation of [P]-orthophosphate (PerkinElmer) into MBP.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For measurement of endogenous BRAF kinase activity, D04 or A375 cells were seeded in 10cm dishes and harvested in 300 μl of NP40 buffer (Table S3) as described above.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Protein concentrations were determined and equal amounts of protein were immunoprecipitated as described above.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For measurement of mutant BRAF kinase activities, COS-7 cells were transiently transfected with myc-tagged BRAF and cells in one 35 mm diameter well were harvested in 200μl of NP40 buffer (Table S3).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The relative concentrations of exogenously expressed RAF in these cell lysates were determined by quantitative western blotting using the myc antibody (Cell Signaling Technology) specified above.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Bands were quantified using the Odyssey infrared imaging system (LI-COR Biosciences).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Equivalent amounts of RAF were immunoprecipitated using rabbit myc antibody (Abcam) as specified above.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Endogenous and transiently transfected RAF proteins were immunoprecipitated in a total of 300 μl NP40 buffer (Table S3) for 2 hr at 4°C and immunoprecipitates were washed sequentially three times with chilled wash buffer (Table S3) containing decreasing concentrations of KCl (1M KCl, 0.1M KCl and no KCl).

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The first-step reaction was initiated by addition of 20 μl MKK buffer (containing GST-MEK and GST-ERK, Table S3) to the beads and incubated at 30°C for 10 min in the case of myc-tagged BRAF or 30 min for endogenous BRAF and CRAF.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Reactions were terminated by the addition of 20 μl KILL buffer (Table S3), which contains EDTA to chelate Mg ions and inhibit kinase activity.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The reaction supernatants were collected from the beads and transferred into fresh tubes for the second step reaction.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

5 μl of supernatant was incubated with 25 μl MBP buffer containing [γ-P]ATP (PerkinElmer) for ten minutes at 30°C in triplicate to measure ERK activity.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The second reaction was terminated by spotting 20 μl of reaction mix onto a 1cm piece of P81 paper (VWR International), which was then dropped into 400ml 25mM orthophosphate solution.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The papers were washed three times in 400 ml 25 mM orthophosphate solution to remove the unincorporated ATP and the [P]-orthophosphate incorporated into MBP was determined using Cerenkov counting.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For transfected samples, the background counts were determined using lysates of cells transfected with the empty vector.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For endogenous protein, samples in which no RAF was immunoprecipitated were used.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Background values were removed and to ensure linearity, assays were used at below 50% saturation.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

To determine BRAF and BRAF sensitivity to 885-A, immunoprecipitated BRAF was preincubated with drug in KCl-free buffer for 10 min at room temperature prior to the first-step reaction.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

To measure the activity of purified BRAF, a 96-well DELFIA-based assay system was used.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Full-length rabbit MEK1 protein was expressed with a GST tag at the N-terminus and a C-terminal histidine tag in Escherichia coli JM109 bacteria and purified by nickel-agarose affinity chromatography.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Full length BRAF protein was generated by infection of SF9 insect cells with a recombinant baculovirus expressing full-length human BRAF with an N-terminal histidine tag and purified as above.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

For the kinase assays, all incubations were at room temperature with shaking.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

4 μg GST-MEK1, 100-200ng purified BRAF and 1 μl inhibitor at the required concentrations (0.001 to 100 μM final concentration) were added to the wells of glutathione-coated plates and preincubated for 10 min.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

ATP in DELFIA assay buffer (20 μL; Table S3), to give a final concentration of 100 μM, was added to each well, and the plates were incubated for 45 min.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The plates were washed 3X with 200 μl 0.1% tween20/water.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

Primary antibody (rabbit anti-phospho MEK1/2 diluted 1/2000, Cell Signaling Technologies) and Eu-labeled anti-rabbit secondary antibody (diluted 1/1000, Perkin-Elmer) were preincubated for 30 min and 100 μl was added to the plates and incubated for a further hour.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The plates were washed as before, and 100 μl DELFIA enhancement solution (Perkin-Elmer, Turku, Finland) was added.

PMC2872605

Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.

The plates were sealed and incubated for 30 min and europium counts measured on Spectramax M5 plate reader (Molecular Devices, Wokingham, UK).