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

Figure 3.

Figure 3

BRAF Binding to CRAF Requires RAS

(A) Myc-epitope tagged CRAF or R89LCRAF (R89L), 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. Myc-tagged CRAF was immunoprecipitated (IP) and the immunocomplexes were western blotted (WB) for endogenous BRAF or myc-CRAF. Endogenous BRAF and myc-CRAF levels in the cell lysates are also shown.

(B) Myc-epitope tagged BRAF or R188LBRAF (R188L) 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. Myc-tagged BRAF was immunoprecipitated (IP) and the immunocomplexes were western blotted (WB) for myc-BRAF or endogenous CRAF. Myc-BRAF and endogenous-CRAF levels in the cell lysates are also shown.

(C) Membrane or cytosol fractions were prepared from untreated (−) or 885-A (1 μM) treated D04 cells. BRAF, CRAF, Tubulin (cytosol control) and HRAS (membrane control) were western blotted in the total lysate (TL), cytosolic fraction (CYT) and membrane fraction (MEM). The graph shows the quantification of the relative levels of BRAF and CRAF in the membrane and cytosol fractions.

(D) PMWK cells were pretreated with DMSO or 885-A (1 μM, 60 min) and then treated with EGF (10 ng/ml) for the times shown in minutes (min). Endogenous CRAF was immunoprecipitated (IP) and the precipitates were western blotted (WB) for BRAF and CRAF. The lysates were also western blotted for BRAF, CRAF, phospho-MEK (ppMEK), phospho-ERK (ppERK) and total ERK2.

(E) D04 cells were treated with DMSO (−) or sorafenib (+; 10 μM) for 4 hr. Endogenous BRAF was immunoprecipitated and the immunocomplexes left untreated or incubated with calf intestinal phosphatase (CIP; 5U, 30°C, 30 min) in the presence or absence of phosphatase inhibitors (P'ase Inh). Immunocomplexes were western blotted for BRAF and CRAF.

(F) D04 cells were treated with DMSO (−), PD184352 (PD; 1 μM) or 885-A (1 μM) for 4 hr. Endogenous CRAF (IP: CRAF) was immunoprecipitated and the immunocomplexes were western blotted (WB) for BRAF or CRAF. BRAF, CRAF, and phospho-ERK (ppERK) levels in the cell lysates are shown.