Hyperactivation of MEK-ERK1/2 occurs predominately through C-RAF. (a) WM1366 cells were transfected using Oligofectamine (Invitrogen, Carlsbad, CA, USA) with 25 nM siRNAs for control, A-RAF, B-RAF and C-RAF (Dharmacon, Lafayette, CO, USA). The siRNA sequences (5′ to 3′) are: control-UAGCGACUAAACACAUCAAUU; A-RAF-ACCGAGAU CUCAAGUCUAA; B-RAF-ACAGAGACCUCAAGAGUAAUU; and C-RAF-GCACGGAGAUGUUGCAGUA. After 72 h, cells were treated with either DMSO (−) or 1 μM PLX4720 for 1 h. Lysates were analyzed by western blot analysis using antibodies for A-RAF, B-RAF, C-RAF (Santa Cruz Biotechnology), (p)MEK, MEK, (p)ERK1/2 and ERK1/2. (b) WM1366 cells were transfected with different combinations of siRNA and processed as in a.