Western Blot Protocol for Protein Analysis

Cells were lysed in pierce IP lysis buffer (Thermo Fisher) and separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE); and after electrophoresis, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). In our lab, 5% bovine serum albumin (BSA) diluted rabbit anti-MCP antibody (1:1,000 dilution) was prepared, in which the patent number was CN111363758A (8 (link)). P-JNK3(1:1,000 dilution), P-p38 mitogen-activated protein kinase (MAPK) (1:1,000 dilution), caspase-3 (1:1,000 dilution), cleaved caspase-3 (1:1,000 dilution), and rabbit anti-β-tubulin antibody (1:2,000 dilution) was purchased from Abcam. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5,000) was purchased from KPL (USA). And then we used the HRP-DAB Chromogenic Substrate Kit (Tiangen, China) to visualize according to the manufacturer’s instructions and took photos.