Fig. 2

The Y35N Mutation Disrupts RHEB-BRAF Interaction Resulting in Increased BRAF/CRAF Heterodimerization and Activation. (a) Top: Western blot for BRAF, CRAF, and FLAG is shown. HEK 293T cells were transfected with plasmids expressing FLAG-RHEB WT, FLAG-RHEB Y35N, or an empty plasmid expressing no protein (Neg). Cell Lysate was collected 48 h post transfection, and an immunoprecipitation (IP) using anti-FLAG antibody was carried out. Bottom: Graph showing the percentage of BRAF bound RHEB Y35N compared to RHEB WT. A BRAF/RHEB ratio was determined for RHEB WT and for RHEB Y35N using ImageJ to calculate the Western blot band intensities of BRAF and FLAG-RHEB as seen in Western blot above. The BRAF/RHEB ratio for RHEB WT was set to 100%, and RHEB Y35N was normalized to RHEB WT. The graph depicts the results from three separate experiments. b Cell lysates were collected from NIH 3T3 cell lines stably expressing RHEB WT or RHEB Y35N. Immunoprecipitation of endogenous BRAF was performed from these lysates. Western blots against CRAF and BRAF are shown. The cell line used for BRAF IP is indicated above the figure as WT (RHEB WT) or Y35N (RHEB Y35N)