Anti tigar

The cochlear explants and HEI-OC1 cells were harvested and lysed with radio-immunoprecipitation assay lysis buffer (Protein Biotechnology, China) containing a protease inhibitor cocktail (Sigma, USA) for 30 min on ice. The lysates were centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was collected, and the protein concentrations were measured by the BCA assay (P0012, Beyotime Institute of Biotechnology). Equal amounts of each protein sample were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Germany). The membranes were blocked with 5% non-fat dried milk in Tris-buffered saline and Tween 20 (TBST) for 1 h at room temperature and then incubated with the primary antibodies in TBST containing 3% non‐fat dried milk at 4 °C overnight. The primary antibodies were anti-TIGAR (1:1000 dilution, Abcam), anti‐cleaved Caspase‐3 (1:500 dilution, CST), anti‐Bax (1:500 dilution, Abcam), anti-Bcl-2 (1:500 dilution, Abcam), anti-p38(1:1000 dilution, CST), anti-p-p38(1:2000 dilution, CST) and anti‐β‐actin (1:1000 dilution; ZSGB‐BIO). The protein signals were visualized using an ECL kit (Millipore, Billerica, MA, USA). Semi-quantification of the western blot results was performed using Image J to measure the intensities of the bands.