Figure S4.

Characterization of ^T529NBRAF and the pan-RAF Inhibitors ZM336372 and RAF265, Relates to Figure 4

(A) ^T529NBRAF is activated by NRAS and KRAS. COS cells were transiently transfected with myc-epitope tagged BRAF, or ^T529NBRAF (T529N) in the presence of ^G12VNRAS or ^G12VKRAS as indicated. BRAF kinase activity was measured in an immunoprecipitation kinase assay. 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 ^G12VNRAS or ^G12VKRAS as appropriate.

(B) Pan-RAF inhibitors drive BRAF binding to CRAF but not ERK activation. DO4 cells were treated with ZM336372 or RAF265 for 4 hr. CRAF (IP: CRAF) was immunoprecipitated and the immnocomplexes were western blotted (WB) for BRAF or CRAF. BRAF and CRAF levels in the cell lysates are shown and the lysates were also western blotted for phosphorylated ERK (ppERK) and total ERK2.