Drug treatment and western blotting

BRAF-mutant A375 and WM2664 cells were treated with BRAF inhibitor vemurafenib (Selleckchem) or ERK inhibitor SCH772984 (Merck, kindly provided by Ahmed Samatar), and NRAS-mutant SK-MEL-119 and Mel224 cells were treated with MEK inhibitor trametinib (Selleckchem) or SCH772984 for 24 and 48 h before samples were collected in RIPA lysis buffer. For PREX1 protein stability experiments, cells were co-treated with cycloheximide (50 μg/ml) and SCH772984 for a 24 h timecourse before samples were collected in RIPA. Whole cell lysates were resolved on 10% SDS-PAGE gels and western blotting was performed using antibodies to phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-RSK (Ser308), phospho-RSK (Thr359/Ser363), total RSK1/2/3, and c-myc (MYC) (Cell Signaling); β-actin and vinculin (Sigma), and PREX1 (6F12) (27). IRDye800-conjugated anti-mouse and anti-rabbit secondary antibodies were from Rockland Immunochemicals.