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.	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.	IC50 values were determined using GraphPad Prism (Graphpad Software Inc., San Diego, CA, USA).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Experiments were performed under Home Office license authority with local Ethics Committee approval.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	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.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Genotyping was performed by PCR using DNA prepared with DNeasy kits (QIAGEN).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	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).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	A+C detects the targeted allele Braf (140 bp).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	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.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	RNA was prepared using the RNEasy kit (QIAGEN).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	First-strand cDNA synthesis was performed with 500ng total RNA and random hexanucleotides (Random Primers, Invitrogen).
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	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.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	For immunohistochemistry (IHC), tumors were fixed in 10% buffered formalin and embedded in paraffin.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Sections (3-10μm) were stained with hematoxylin and eosin using standard protocols.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	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.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	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.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	Immunohistochemical staining was analyzed by two of the authors on a multi-headed microscope.
PMC2872605	Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF.	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.

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.

IC50 values were determined using GraphPad Prism (Graphpad Software Inc., San Diego, CA, USA).

PMC2872605

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

Experiments were performed under Home Office license authority with local Ethics Committee approval.

PMC2872605

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

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.

PMC2872605

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

Genotyping was performed by PCR using DNA prepared with DNeasy kits (QIAGEN).

PMC2872605

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

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).

PMC2872605

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

A+C detects the targeted allele Braf (140 bp).

PMC2872605

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

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.

PMC2872605

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

RNA was prepared using the RNEasy kit (QIAGEN).

PMC2872605

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

First-strand cDNA synthesis was performed with 500ng total RNA and random hexanucleotides (Random Primers, Invitrogen).

PMC2872605

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

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.

PMC2872605

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

For immunohistochemistry (IHC), tumors were fixed in 10% buffered formalin and embedded in paraffin.

PMC2872605

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

Sections (3-10μm) were stained with hematoxylin and eosin using standard protocols.

PMC2872605

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

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.

PMC2872605

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

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.

PMC2872605

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

Immunohistochemical staining was analyzed by two of the authors on a multi-headed microscope.

PMC2872605

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

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.