Ab240220

Protein extraction was performed on ice, and the reaction solution was pre-cooled with ice. The tissues or cells were washed with cold phosphate-buffered saline (PBS), lysed with radioimmunoprecipitation assay (RIPA) (Beyotime Institute of Biotechnology) lysis buffer, centrifuged (at 10,000 x g for 15 min at 4̊C), discarded, and resuspended in a cell lysate containing phosphatase inhibitor or protease inhibitor (Beyotime, Shanghai, China). Bicinchoninic acid (BCA) protein concentration assay kit (Beyotime Institute of Biotechnology) was used for the determination of protein quality. Then, the protein was added to the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membranes (EMD Millipore). After 5% milk blocking, membranes were coated with primary antibodies against: Collagen II (ab34712), ATF4 (ab31390), CHOP (ab240220), cleaved caspase-9 (ab2324), and β-actin (as loading control, ab179467) overnight at 4˚C. All the antibodies were purchased from Abcam. Membranes were then incubated with secondary antibody for 1 h at room temperature. Chemiluminescent ECL substrate (Beyotime Institute of Biotechnology) was used to expose the band.