PMCID
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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IC50 values were determined using GraphPad Prism (Graphpad Software Inc., San Diego, CA, USA).Transgenic MiceExperiments were performed under Home Office license authority with local Ethics Committee approval.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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To activate CreERT2, four doses of tamoxifen (Sigma; 10mg each in 100% ethanol) were applied topically to the shaven skin on the backs of the mice every other day for 7 days.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Genotyping was performed by PCR using DNA prepared with DNeasy kits (QIAGEN).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Braf and Braf were analyzed using primers: A) 5′-GCCCAGGCTCTTTATGAGAA-3; B) 5′-AGTCAATCATCCACAGAGACCT-3′; and C) 5′-GCTTGGCTGGACGTAAACTC-3′. A+B detects the wild-type BRAF allele (466 bp product) and Braf, the Cre-recombinase recombined allele (518pb product).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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A+C detects the targeted allele Braf (140 bp).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Tyr::CreERT2 was analyzed using primers 5′-GAAGCAACTCATCGATTG-3′ and 5′-TGAAGGGTCTGGTAGGATCA-3′. Kras was analyzed using primers 5′-CGCAGACTGTAGAGCAGCG-3′ and 5′-CCATGGCTTGAGTAAGTCTGC-3′.mRNA expression analysis was performed by RT-PCR.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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RNA was prepared using the RNEasy kit (QIAGEN).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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First-strand cDNA synthesis was performed with 500ng total RNA and random hexanucleotides (Random Primers, Invitrogen).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Tyrosinase (Tyr), was detected using primers 5′-TGGTTCCTTTCATACCGCTC-3′ and 5′-CAGATACGACTGGCTTGTTCC-3′; Dct with 5′-GCAAGATTGCCTGTCTCTCC-3′ and 5′-AGTCCAGTGTTCCGTCTGCT-3′; Pax3 with 5′-CCAGGATGATGCGGCCCGGCCCGGG-3′ and 5′-AGGATGCGGCTGATAGAACTCACTG-3′; and silver/gp100 (Si) with 5′-GGAGAGGTGGCCAGGTATC-3′ and 5′-CAGTAATGGTGAAGGTTGAAC-3′. The control Gapdh was detected with 5′- GATGGCCCCTCGGAAAGCT-3′ 5′-CCAGTGAGCTTCCCGTTCAGC-3′. To sequence Kras cDNA, a 238 bp product from Kras cDNA was PCR amplified using primers 5′-GGCGGCAGCGCTGTGGCGGCG-3′ and 5′-CGTAGGGTCATACTCATCCAC-3′ and directly sequenced using these primers and automated dideoxy sequencing.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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For immunohistochemistry (IHC), tumors were fixed in 10% buffered formalin and embedded in paraffin.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Sections (3-10μm) were stained with hematoxylin and eosin using standard protocols.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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For S100 and Ki67 staining, antigen retrieval was performed in citrate buffer (pH 6.0, 30 min) and revealed using a rabbit polyclonal antibody (Dako, 1/1000), the Rabbit Envision Peroxidase kit and the AEC substrate chromogen (Dako) for S100, and a rat monoclonal antibody (Dako,1/25), the rat Vectastain ABC kit (Vector Labs, USA) and DAB as chromagen for Ki67.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Positive (a well characterized sample of mouse melanoma) and negative (omission of the primary antibody and substitution with preimmune serum) controls were included in each slide run.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Immunohistochemical staining was analyzed by two of the authors on a multi-headed microscope.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Tumor cell lines were established by mechanically dissociating tumors in DMEM/20%FCS/Primocin (0.1mg/ml - InvivoGen) and clonal lines were selected by limiting dilution.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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For 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).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Calf intestinal phosphatase (CIP) was from New England Biolabs (NEB).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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PD184352, sorafenib and PLX4720 were synthesized in-house; 885-A was synthesized by Evotec AG (Abingdon, UK).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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All drugs were prepared in DMSO.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Synthetic routes are available on request.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The 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).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Briefly, the vector backbone is pUC19 and the elongation factor 1α (EF1α) promoter is used to drive exogenous protein expression.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The vector includes the first intron from human EF1α to assist mRNA processing during expression.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The vector introduces an amino-terminal myc-epitope tag (EQKLISEEDL) onto the exogenously expressed protein.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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In addition, a version of this vector was used that incorporates a FLAG (DYKDDDKGS), rather than a myc-epitope tag.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Human 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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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African green monkey kidney COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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All cell lines were incubated at 37°C and 10% CO2.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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For inhibitor treatment, the drugs were dissolved in DMSO and added to the medium for 2-5 hr.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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When two compounds were used, the first was added 30 min prior to the second.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The amount of growth (% DMSO controls) was determined using sulphorhodamine B reagent (Monks et al., 1991) as follows.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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1,000-10,000 cells (depending on cell type) were plated into 96-well plates in 100 μL medium.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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%).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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After rinsing, the bound stain was disolved using 100 μL 10 mM Tris (pH 8.0) and the absorbance at 540 nm determined.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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3 × 10 D04 cells per 35 mm diameter well were seeded in 2ml growth medium the day before transfection.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The mix was vortexed for 10 s and incubated for 5-10 min before adding the complexes dropwise to the cells.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The cells were serum-starved the day after transfecting and extracts were prepared 48 hr after transfection.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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For transient protein expression in D04 cells, Lonza Nucleofector Technology (Lonza, Cologne AG) was used.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The cells were re-plated into 35mm diameter tissue culture wells and incubated for 48 hr before preparation of cell extracts.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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For generation of stable lines, D04 cells were transfected using Effectene (Invitrogen) and selected in G418.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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3-4x10 cells were plated in 35 mm diameter wells and incubated overnight.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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10 μl Effectene reagent was added and the mixture was incubated for another 5-10 min.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The cells were washed with 2ml PBS and 1.6ml fresh serum containing medium was added.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The DNA complexes were diluted with 600 μl of culture medium and the mixture added to the cells drop-wise.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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After six hours, the medium was replaced with 2ml of fresh growth medium.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The medium was refreshed weekly and after 2-3 weeks, single colonies were selected and expanded.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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For transient expression in COS-7 cells, Lipofectamine (Invitrogen) was used.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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2x10 cells were plated into 35mm diameter wells and incubated overnight.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The cells were washed twice with 1ml serum-free DMEM, and then overlaid with 800 μl of serum free DMEM.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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200 μl of serum free DMEM was added to the DNA:Lipofectamine mix, and the total volume was added to the cells.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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After six hours, the complexes were removed and replaced with 2ml of normal culture medium.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Cell extracts were prepared two days following transfection.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Culture medium was aspirated from cells and cells were placed on ice and washed three times in ice-cold PBS.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Immunoprecipitations were performed in 300 μl cell lysates from one 35mm diameter well for endogenous protein or from 2-3 wells for transfected protein.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Specific bands were detected using fluorescent-labeled secondary antibodies (Invitrogen; Li-COR Biosciences) and analyzed using an Odyssey Infrared Scanner (Li-COR Biosciences).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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For CIP treatment, immunoprecipitates were washed twice with NP40 lysis buffer (Table S3) and once in CIP buffer (Table S3).
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Controls were incubated in CIP buffer without CIP.
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PMC2872605
|
Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The reactions were performed at 30°C for 30 min before analysis on SDS-PAGE.
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PMC2872605
|
Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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D04 cells were plated on two 10cm dishes per treatment and grown to confluency.
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PMC2872605
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Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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).
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PMC2872605
|
Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Cells were disrupted by passing them through a 9G syringe ten times, followed by another ten times through a 19G syringe (Terumo Medical).
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PMC2872605
|
Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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Lysates were centrifuged at 900 × g for five minutes to pellet the nuclear proteins.
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PMC2872605
|
Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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The supernatant was transferred to fresh 1.5ml ultracentrifuge tubes (Beckman Coulter) and 200 μl removed as a total lysate control.
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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.
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PMC2872605
|
Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
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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.
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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.
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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.
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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.
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PMC2872605
|
Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.
|
The 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.
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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.
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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.
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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.
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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).
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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.
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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).
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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.
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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).
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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.
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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.
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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.
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