Western Blot Analysis of Cellular Proteins

As described in (37 (link)), for whole-cell extracts, cells were lysed in 50 mM NaCl, 1.0% IGEPAL CA-630, and 50 mM tris-HCl (pH 8.0). Buffer was supplemented with cOmplete (Roche, #4693132001) and PhosSTOP (Roche, #4906845001). Protein concentration was determined by a Qubit protein assay reagent (Thermo Fisher Scientific, #Q33212). Samples were denatured at 95°C for 5 min in a Roti-Load reducing buffer (Carl Roth, #K929.1) before SDS–polyacrylamide gel electrophoresis using NuPAGE bis-tris gels and then transferred to 0.45 μM polyvinylidene difluoride membranes. Membranes were blocked for 30 min with 5% milk in PBS with 0.3% Tween 20 (PBST 3%). Membranes were washed twice with PBST 3%. The membrane was incubated with primary antibodies against MOF (1:1000; Bethyl, #A300-992A), actin (1:10,000; Sigma-Aldrich, #A2066), H3 (1:5000; Active Motif, #39763), p16^INKA (1:1000; Abcam, #ab54210), p19^INKD (1:1000; Abcam, #ab80), and H4K16ac (1:2000; Millipore, #07-32) diluted in PBS–5% BSA. The membrane was then incubated with PBS–5% BSA containing horseradish peroxidase–conjugated anti-mouse (GE Healthcare, #NA931-1ML) or anti-rabbit (GE Healthcare, #NA934) (1:10,000).