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. 2010 Jan 22;140(2):209–221. doi: 10.1016/j.cell.2009.12.040

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© 2010 ELL & Excerpta Medica.

Open Access under CC BY 3.0 license

PMC Copyright notice

BRAF and Not CRAF Inhibition Drives CRAF Binding to BRAF and CRAF Activation

(A) COS cells were transiently transfected with myc-epitope tagged BRAF, or ^T529NBRAF (T529N) in the presence of ^G12VHRAS (RAS) and their kinase activity was measured. The data represent one assay performed in triplicate, with error bars to represent standard deviations from the mean. Activity (%) is relative to wild-type BRAF activated by ^G12VHRAS.

(B) As in (A) but immunocomplexes were treated with DMSO (−) or 885-A for 10 min prior to measuring their kinase activity. The data represent one assay performed in triplicate, with error bars to represent standard deviations from the mean. Activity (% control) is relative to the untreated kinase.

(C) Myc-epitope tagged BRAF, ^T529NBRAF (T529N), or an empty vector control (EV) were transfected into D04 cells. After 48 hr the cells were treated with DMSO (−) or 885-A (1 μM) for 4 hr. The endogenous CRAF was immunoprecipitated and the immunocomplexes were western blotted for myc-BRAF or endogenous CRAF. Myc-BRAF and endogenous CRAF levels in the cell lysates are also shown.

(D) Myc-epitope tagged BRAF, ^D594ABRAF (D594A), or an empty vector control (EV) were transfected into D04 cells. After 48 hr the myc-BRAF was immunoprecipitated (IP) and the immunocomplexes were western blotted for mycBRAF and endogenous CRAF. Myc-BRAF and endogenous CRAF levels in the cell lysates are also shown.

(E) Myc-epitope tagged BRAF, ^D594ABRAF (D594A), or an empty vector control (EV) were transfected into D04 cells. After 48 hr the cells were treated with DMSO (−), sorafenib (SF; 10 μM) or 885-A (1 μM) for 4 hr. The cells extracts were western blotted for myc-BRAF, phospho-MEK (ppMEK), phospho-ERK and total ERK2 (loading control). Note that ERK2 runs as a doublet due to the separation of the phosphorylated and nonphosphorylated protein.

(F) Myc-epitope tagged BRAF, ^K483MBRAF (K483M), ^D594VBRAF (D594V), ^D594ABRAF (D594A), or an empty vector control (EV) were transfected into D04 cells. After 48 hr, the cells were treated with DMSO (−) or sorafenib (SF; 10 μM) for 4 hr. Cell extracts were blotted for myc-BRAF, phospho-MEK (ppMEK), phospho-ERK (ppERK) and CRAF (loading control).

(G) D04 cells were treated with sorafenib (10 μM) for various times and CRAF kinase activity was measured. Data is for one assay performed in triplicate, with error bars to represent standard deviations from the means. Inset: D04 cells were treated with sorafenib (SF) for 4 hr and CRAF was immunoprecipitated and western blotted for S338 phosphorylation (pS338). CRAF levels in the lysate are shown as a loading control.

(H) D04 cells stably expressing flag-epitope tagged CRAF (CRAF) or ^T421NCRAF (T421N) were treated with DMSO (−), PD184352 (PD; 1 μM), sorafenib (SF; 10 μM) or 885-A (1 μM) for 4 hr. The flag-CRAF was immunoprecipitated (IP) and the immunocomplexes were western blotted for endogenous-BRAF or flag-CRAF. Endogenous BRAF, flag-CRAF and phosphorylated ERK (ppERK) levels in the cell lysates are also shown.