Anti p38 mapk

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.

One hundred microliters of RIPA lysate was added to 20 mg of tissue, homogenized on ice, centrifuged at 12,000 rev/minute for 10 minutes at 4°C, and the supernatant was separated to determine the protein concentration. The protein was separated by 10% SDS-PAGE electrophoresis with a loading of 40 µg in a volume of 20 µL. After the electrophoresis, a “sandwich” was prepared, and the protein was electrotransferred to the PVDF membrane. After blocking with 5% nonfat dry milk for 2 hours, added anti-uPA (1:600), anti-p38MAPK (1:500), and mouse anti-β-actin (clone AC-15; 1:10,000, Sigma-Aldrich). After washing the membrane, added the secondary antibody, incubated for 50 minutes at room temperature on the shaker. At the end, the membrane was washed five times with TBST for 5 minutes each time, and the ECL was exposed. Gelpro 32 analysis was performed for gel image analysis.