Supersignal ecl system

Cells were lysed with cold RIPA buffer plus complete protease inhibitor cocktail (Roche Applied Science). The signal was detected using the SuperSignal ECL system (Thermo Scientific). The following antibodies were used for immunoblotting: SOD1 and Nrf2 (Santa Cruz Biotechnology) and CAT, HMOX-1, pan-phospho, and β-actin (Cell Signaling Technologies).

Cells were lysed in Laemmli buffer, and the protein extracts were tested by antibody specific for IRF4 diluted to 1:1,000; secondary antibodies were used at 1:10,000. Anti-rabbit antibody, conjugated to HRP (GE Healthcare Life Sciences, Little Chalfont Buckinghamshire, UK), was used as a secondary antibody. The SuperSignal ECL system was used for probing target proteins (Thermo Fischer Scientific). After the membranes had been probed for the target protein, they were stripped and re-probed for β-actin.

For western blot analysis, mouse myocardial tissues from infarcted area of TAC groups and anterior left ventricular of sham operated groups were homogenized in radio-immunoprecipitation assay buffer (Beyotime, Shanghai, China) and protein concentration was determined using the BCA protein assay kit (Beyotime). Aliquots containing 20 μg of protein were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to nitrocelullose membranes (Pall Corporation, USA). Membranes were incubated in 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature, followed by incubation in primary antibodies at 4°C overnight. The primary antibodies used and their dilutions were as follows: anti-SIRT6, 1:2,000; anti-α-tubulin, 1:2,000; anti-TERT, 1:1,000; anti-TRF1, 1:1,000; anti-collagen I, 1:5,000; anti-FN, 1:1,000; anti-α-tubulin, 1:1,000; β-actin, 1:2,000 (all from Abcam, Cambridge, United Kingdom). After washing in TBST, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Signal detection was performed using the SuperSignal ECL system (ThermoScientific, Waltham, Massachusetts) and bands were analyzed by ImageJ software. Band intensity was normalized to that of α-tubulin or β-actin.