Quantitative Western Blot Analysis

The protein concentrations within the fractionated sample loaded onto the gel were normalized by total protein content using the bicinchoninic acid (BCA) assay (Thermo Scientific Pierce, Waltham, MA). Proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Gels were rinsed in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol), and the proteins transferred onto a nitrocellulose membrane in transfer buffer for 1.5 h at 100 V at 4°C. After transfer, membranes were blocked with 5% milk in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.1% Tween) for 1 h and washed with PBST four times for 5 min each time. Primary rabbit antiacetyllysine antibody (Cell Signaling, Danvers, MA) was diluted 1,000-fold in 5% bovine serum albumin (BSA), added to the membranes, and incubated in the cold room with shaking. The membrane was washed 4 times with PBST for 5 min each time and incubated for 1 h in the dark at room temperature with anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling, Danvers, MA) diluted 2,000-fold in 5% milk. The membrane was washed 4 times with PBST for 5 min each time, incubated in ECL blotting substrate (Abcam), and imaged in the Protein Simple machine (Bio-Techne) (13 (link), 19 (link)).