Mouse anti β actin

Following the aforementioned treatments, H9c2 cells were washed thoroughly with ice-cold PBS solution, and RIPA buffer (Beyotime Institute of Biotechnology) was then added to the wells and incubated for 30 min on ice. Protein was quantified using a BCA assay. A total of 30 µg proteins/lane were separated by 8% SDS-PAGE and transferred to PVDF membranes at 4˚C and 200 mA for 2 h. The membranes were blocked by 5% skimmed milk powder in TBS with Tween-20 (TBS-T) solution for 2 h at room temperature and then incubated at 4˚C overnight with the following primary antibodies: Anti-CHOP (1:1,000; cat. no. DF6025; Affinity Biosciences), anti-GRP78 (1:1,000; cat. no. AF5366; Affinity Biosciences), anti-caspase-12 (1:1,000; cat. no. AF5199; Affinity Biosciences) and mouse anti-β-actin (1:1,000; cat. no. T0022; Affinity Biosciences). A horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. 111-095-003; Jackson ImmunoResearch Laboratories, Inc.) was then added for 2 h at room temperature after the membranes were washed five times with TBS-T buffer. Finally, the membranes were washed with TBST, and signals were visualized with an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology). The protein band densities were quantified with ImageQuant TL software (version 7.0; Cytiva).

Following the treatments described in previous procedures, H9c2 cells were washed well with an ice-cold PBS solution, and then, we added a RIPA solution (Beyotime, China) to the wells and incubated the cells for 30 min on ice. Subsequently, the supernatants were collected after centrifugation of the lysates. The BCA method was used to measure the protein concentration. The proteins were separated by SDS-PAGE and transferred to PVDF membranes at 4 °C and 200 mA for 2 h. Then, the membranes were blocked in a TBST solution for 2 h at room temperature and incubated at 4 °C overnight with the following primary antibodies: anti-dual phospho-p38 MAPK (Thr¹⁸⁰ and Tyr¹⁸²) (Sigma, 1:1000), anti-p38 MAPK (Sigma, 1:1000), anti-CHOP (Affinity, 1:1000), anti-GRP78 (Affinity. 1:1000), anti-caspase12 (Affinity, 1:1000) and mouse anti-β-actin (Affinity, 1:1000). A secondary antibody conjugated to horseradish peroxidase (Jackson, 1:2000) was added and incubated for 2 h at room temperature. Finally, the membranes were washed in TBST, and the signals were visualized with an enhanced chemiluminescence detection kit (ECL, Beyotime, China). The density of the protein bands was quantified by IQuantTL (GE Healthcare, USA). Relative protein expression was calculated with normalization to β-actin expression.