3.2.4. Western Blot Analysis

To measure the capability of our compounds to interfere with BRAF pathway, pre-treated A375 and Colo205 cells were lysed. We then performed protein analysis through Western blot by using the following primary antibodies: anti-phospho ERK 1/2 (clone E10; dilution 1:1000; Cell Signaling Technology, Danvers, MA, USA) and anti-ERK (clone 137F5; dilution 1:1000; Cell Signaling Technology); anti-phospho MEK (clone S217/221; dilution 1:1000; Cell Signaling Technology); anti-MEK1,2 (clone 9122; dilution 1:1000; Cell Signaling Technology); anti-BRAF v600e (dilution 1:1000; Abcam, Cambridge, UK); and anti-total BRAF (dilution 1:1000; ThermoFisher, Waltham, MA, USA). Vinculin (clone E1E9B; dilution 1:1000; Cell Signaling Technology) was used as a loading control. The total protein amount was determined using the bicinchoninic acid (BCA) protein assay reagent (Thermo Scientific, Waltham, MA, USA). Equivalent amounts of protein were separated by SDS–PAGE on precast Bolt 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA). Proteins were then transferred from gel to nitrocellulose with a Trans-Blot Turbo transfer system (Biorad, Hercules, CA, USA), probed with goat anti-rabbit IgG (H+L) secondary antibody, HRP conjugate (Invitrogen) or goat anti-mouse IgG (H+L) secondary antibody, HRP conjugate (Jackson ImmunoResearch Inc., Langcaster, PA, USA), and detected using enhanced chemiluminescence technique (Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate; Perkin Elmer, catalog # NEL105001EA, Waltham, MA, USA).