Anti h3k56ac

Protein extracts from hepatocytes or liver tissues were made in the lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA and freshly added 1 mM PMSF and an additional protease cocktail tablet from Roche at one tablet/10 mL final buffer volume). Protein extracts were resolved on an SDS-PAGE gel and transferred to nitrocellulose membranes. Immunoblots were blocked in TBST with 5% skimmed milk for 1 hour at RT and incubated overnight at 4°C with the following primary antibodies: anti-NAMPT, anti-Actinin (Proteintech), anti-H3K9Ac, anti-H3K56Ac, anti-histone H3 (Cell Signaling Technology), anti-SIRT1 (Santa Cruz Biotechnology), anti-SIRT6 (Abcam). Following 3 washes in TBST, immunoblots were incubated with HRP-conjugated secondary antibody for 1 hour. After 3 washes in TBST, the immune complexes were detected using the ECL detection reagents (Sigma).

Protein lysate were prepared in RIPA lysis buffer (Pierce, Rockford, USA). 30 μg of total proteins were separated by SDS-PAGE, and transferred onto PVDF membrane (Pierce). After blocking with 5% non-fat milk, the blots were incubated with primary antibody at 4 °C overnight. The blots were incubated with HRP-conjugated secondary antibody (Invitrogen). The signal was detected using ECL substrate (Pierce). The following antibodies were used in this study: anti-TXNIP (#14715; 1:1000), anti-NLRP3 (#15101; 1:1000), anti-ASC (#13833; 1:1000), anti-cleaved caspase-1 (#4199; 1:1000), anti-eNOS (#32027; 1:1000), anti-H3K9ac (#9649; 1:1000), anti-H3K56ac (#4243; 1:1000), anti-Histone H3 (#4499; 1:2000) and anti-β-actin (#3700; 1:1000) antibodies were from Cell signaling technologies (CST, Beverly, MA, USA). Anti-SIRT6 (ab191385; 1:2000), anti-H3 (ab1791; 1:2000) and anti-VEGF (ab46154; 1:1000) antibodies were from Abcam (Cambridge, UK).