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. Author manuscript; available in PMC: 2014 Mar 13.
Published in final edited form as: Cancer Cell. 2011 Dec 13;20(6):715–727. doi: 10.1016/j.ccr.2011.11.004

Figure 2. Imatinib, nilotinib and dasatinib drive paradoxical activation of BRAF and CRAF in RAS mutant cells.

Figure 2

A. RAF kinase assays. Myc-epitope tagged BRAF, CRAF, BRAFT529N or CRAFT421N were transiently expressed in COS cells. BRAF and BRAFT529N were co-expressed with HRASG12V, and CRAF and CRAFT421N were co-expressed with HRASG12V and SRCY527F. The myc-tagged proteins were captured and their kinase activity was determined in the presence of imatinib (10μM), nilotinib (1μM) or dasatinib (5μM). The results are the mean of triplicate determinations and error bars to represent SD and are presented relative to DMSO-treated controls (dotted line).

B. Western blots for endogenous BRAF and CRAF in CRAF immunoprecipitates (CRAF IP) or cell lysates from D04 cells treated with DMSO (-), imatinib, nilotinib or dasatinib at the indicated concentrations.

C. Western blots for endogenous BRAF and CRAF in CRAF immunoprecipitates (CRAF IP) or cell lysates from SW620, H460 and Panc1 cells treated with DMSO (-), imatinib (10μM), nilotinib (1μM) or dasatinib (5μM).

D. Western blots for myc-tagged BRAF, endogenous CRAF or tubulin (loading control) in myc immunoprecipitates (myc IP) or cell lysates from D04 cells expressing empty vector control (EV), myc-BRAF (BRAF) or myc-BRAFR188L (R188L) and treated with DMSO (-) or nilotinib (N; 1μM).

E. Western blots for myc-tagged CRAF, endogenous BRAF or tubulin (loading control) in myc immunoprecipitates (myc IP) or cell lysates from D04 cells expressing empty vector control (EV), myc-CRAF (CRAF) or myc-CRAFR89L (R89L) and treated with DMSO (-) or nilotinib (N; 1μM).

F. Western blots for myc-tagged CRAF, HA-tagged CRAF or tubulin (loading control) in myc immunoprecipitates (myc IP) or cell lysates from D04 cells expressing empty vector control (EV), myc-CRAF or HA-CRAF and treated with DMSO (-) or nilotinib (+; 1μM).

G. Western blots for myc-tagged BRAF, HA-tagged BRAF or tubulin (loading control) in myc immunoprecipitates (myc IP) or cell lysates from D04 cells expressing empty vector control (EV), myc-BRAF or HA-BRAF and treated with DMSO (-) or nilotinib (+; 1μM).

H. Western blots for FLAG-tagged BRAF or FLAG-tagged BRAFT529N (FLAG-BRAF), endogenous CRAF, phospho-MEK (ppMEK), phospho-ERK (ppERK) and tubulin (loading control) in CRAF immunoprecipitates (CRAF IP) or cell lysates from D04 cells expressing empty vector control (EV), FLAG-tagged BRAF (BRAF) or FLAG-tagged BRAFT529N (T529N) and treated with DMSO (-), imatinib (I; 10μM), or nilotinib (N; 1μM).

I. Western blots for HA-tagged BRAF (HA-BRAF), myc-tagged BRAF (myc-BRAF) or tubulin (loading control) in myc immunoprecipitates (myc IP) or cell lysates from D04 cells expressing myc-BRAF, myc-BRAFT529N (myc-T529N) or HA-BRAF and treated with DMSO (-), or nilotinib (N; 1μM).

J. Western blots for myc-tagged CRAF (myc-CRAF), FLAG-tagged CRAF (FLAG-CRAF) or tubulin (loading control) in FLAG immunoprecipitates (FLAG IP) or cell lysates from D04 cells expressing myc-CRAF, FLAG-CRAF or FLAG-CRAFT421N (FLAG-T421N) and treated with DMSO (-), or nilotinib (N; 1μM).