Western Blot Analysis of Protein Markers

Total protein was extracted using radio immunoprecipitation assay (RIPA) lysis buffer with freshly added protease inhibitor (Roche, Basel, Switzerland). Western blot analysis was performed as described previously^{17 (link)}. Briefly, 40 μg total protein extract was loaded into a 10% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) followed by transfer onto a polyvinylidene difluoride (PVDF) membranes (Roche, Basel, CH). Membranes were blocked with 5% skim milk, incubated first with rabbit polyclonal antibodies against human SET8 (1:1000; Abcam, Cambridge, UK), p53 (1:2,000; Santa Cruz, CA), anti-p53K382me1(1:1000; Affinity Biosciences, Cincinnati, OH)^{11 (link)}, or β-actin (1:10,000; Santa Cruz, CA) overnight at 4 °C, followed by incubation with secondary HRP-conjugated anti-rabbit IgG antibody (1:5000; Thermo Fisher, New York, NY). Proteins were visualized with an enhanced chemiluminescence reagent (Thermo Fisher, New York, NY) using the FluorChem® HD2 protein imprinting imaging system (Alpha InnoTec, San Leandro, CA).