Effects of KSR1 on Inhibitor-induced ERK Cascade Activation
(A) KSR−/− and WT-KSR1 MEFs were treated with L779450 (10 μM for 1 hr). The catalytic activity of endogenous B-Raf and C-Raf proteins was measured in immune-complex kinase assays using kinase-inactive MEK as a substrate. Lysates were analyzed for pERK and tubulin (loading control) levels. (B) A549, HMCB, and A375 cells expressing either the pLKO.1 vector or pLKO.1-KSR1 shRNA were treated as indicated for 1hr. Lysates were analyzed for pERK and tubulin levels. Depletion of KSR1 is also shown. (C) Inhibitor-induced Raf dimerization occurs at the plasma membrane and is Ras-dependent (Left). Inhibitor-induced KSR1/B-Raf complexes can form in the cytosol, thus inhibiting the formation of C-Raf/B-Raf dimers at the membrane (Right).