Cells were plated at reduced FBS concentration and were exposed to inhibitors at 1 μM or 0.2% DMSO. Lysates were quantified and 20 μg total protein loaded onto 10–12% Mini-PROTEAN TXG Precast Gels (Bio-Rad, USA). Primary antibodies were obtained from Cell Signalling Technology (USA) and the following clones and dilutions were used: P-ERK [Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) #9101, 1:1000] and GAPDH [GAPDH (D16H11) XP (R) #5174, 1:1000]. The P-ERK antibody has been previously cited for studying downstream effects of BRAF inhibitors on MAPK pathway activity [3 (link)], and in-house validation of dilutions was performed. Blots were probed with appropriate secondary HRP-conjugated antibodies and detected using chemiluminescence on a Fusion FX6XT digital imaging system (Vilber Lourmat, Germany).
Densitometry was performed for P-ERK and GAPDH for each cell line at each time point. All experiments were conducted in triplicate. Two-way ANOVA with Tukey’s post-test was performed on densitometry values, again in GraphPad Prism Software.
Densitometry was performed for P-ERK and GAPDH for each cell line at each time point. All experiments were conducted in triplicate. Two-way ANOVA with Tukey’s post-test was performed on densitometry values, again in GraphPad Prism Software.