Trans blot turbo transfer system and reagents

Protein was harvested using a Cell Lysis Buffer (Abcam, Cambridge, UK) with a 2% v/v Halt™ Protease Inhibitor Cocktail (ThermoFisher Scientific). Western blots were performed using Bio-Rad mini-Protean TGX Gels and the Trans-Blot^® Turbo Transfer System and reagents (Bio-Rad, Watford, UK). The antibodies used for blotting were rabbit-anti-AKT1, with mouse-anti-GAPDH as the loading control (both Proteintech, Manchester, UK). The membranes were blocked in 5% milk diluted in TBS-T (0.05%), followed by incubation in secondary antibody (goat anti-rabbit IgG-HRP (1:5000) or goat anti-mouse IgG-HRP (1:5000), both Proteintech). The membrane was incubated in enhanced chemiluminescent reagent (ThermoFisher Scientific) and the signal was detected on a G:BOX F3 imaging system (Syngene, Cambridge, UK). At least three biological replicates per experiment were conducted.

Protein was extracted using Cell Lysis Buffer (Abcam, Cambridge, UK) with 2% v/v protease inhibitor (ThermoFisher Scientific). Western blots were performed using Bio-Rad mini-Protean TGX Gels and Trans-Blot^® Turbo Transfer System and reagents (Bio-Rad, Watford, UK). Antibodies used for blotting were rabbit-anti-MITF, with mouse-anti-GAPDH as loading control (both Proteintech, Manchester, UK). Membranes were blocked in 5% milk diluted in TBS-T (0.05%), followed by incubation in the appropriate secondary antibody (goat anti-rabbit IgG-HRP (1:5000) or goat anti-mouse IgG-HRP (1:5000), both Proteintech). Luminescence was revealed by incubation with enhanced chemiluminescent reagent (ThermoFisher Scientific) and signal detected on a G:BOX F3 imaging system (Syngene, Cambridge, UK). At least four biological replicates per experiment were conducted.