Immunoblot Analysis of Histone Modifications and Cytoskeletal Proteins

Mammalian Cell Lysis buffer (Sigma Aldrich) was used for C2C12 cells and COS7 cells. Immunoblot analysis were prepared as previously described^{15 (link)}. The following antibodies were used: anti-Histone H3 (1:10,000 dilution; ab1791, Abcam, Cambridge, UK), anti-Histone H3 acetyl K9 (1:2,000 dilution; ab4441, Abcam), anti-SIRT1 (1:1000 dilution; ab110304, Abcam), anti-CTTN (1:200 dilution, H-191, Santa Cruz Biotech, Texas, USA), anti-acetylated CTTN (1:50 dilution, 09-881, Millipore, Massachusetts, USA), anti-GFP (1:2,000 dilution, ab13970, Abcam), Anti-DYKDDDDK tag (Flag; 1:10,000 dilution, FUJIFILM Wako Pure Chemicals), anti-GAPDH (1:2000 dilution; MAB374, Sigma Aldrich), anti-β-Actin (1:10,000 dilution; 281-98721, FUJIFILM Wako Pure Chemicals) and anti-Vinculin antibody (1:10,000 dilution; V9131, Sigma-Aldrich). Following antibody was used for immunoprecipitation; anti-Flag antibody-conjugated beads (50 μl for each sample, FUJIFILM Wako Pure Chemicals). The immunoprecipitates were washed three times with TBST and analyzed by immunoblotting. For phalloidin pull-down, the lysate was incubated overnight at 4 °C with 5 units of Biotin-XX Phalloidin (B7474, Thermo Fisher Scientific), then incubated with 500 µg of Dynabeads M-280 Streptavidin (11205D, Thermo Fisher Scientific) for 1 h and washed three times with TBST. The pull-downs were analyzed by immunoblotting.