Immunoblotting

Lysates were isolated using standard protocols from A375, DM440, and DM443 cells expressing the indicated transgenes or A375 cell lines grown on poly-hema coated plates (to promote anchorage independent growth) treated with or without either a fixed concentration of TTM (100 nM), vemurafenib (12.5 nM), or trametinib (300 pM) or increasing concentrations of TTM (25 nM, 50 nM, and/or 100 nM), vemurafenib (3.125 nM, 6.25 nM, and/or 12.5 nM), or trametinib (75 pM, 150 pM, and/or 300 pM) for 7 days. Equal quantities were resolved by SDS–PAGE and immunoblotted as previously described (16) with mouse anti-ßACTIN, rabbit anti-phospho(Thr 202/Tyr 204)-ERK1/2, mouse anti-ERK1/2, mouse anti-HA, rabbit anti-phospho(Ser 235/236)-S6, or mouse anti-S6 primary antibody (Cell Signaling Technology, catalog # 3700, 9101, 9107, 4858, and 2317), followed by detection with either a goat anti-mouse IgG or a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, catalog # 7074 and 7076), and visualized using enhanced chemiluminescence detection reagents (Cell Signaling Technology). The fold change in the ratio of phosphorylated protein to ßACTIN loading control was determined by boxing the appropriate bands of phosphorylated and ßACTIN per representative image with the rectangular selection tool and calculating the total volume of the band in pixels using the Image Lab™ Software (Bio-Rad). The total volume of the phosphorylated protein band in pixels was then normalized to the total volume of the ßACTIN protein band in pixels.