Nbp2 37740

Protein expression of matrix-related molecules (aggrecan and collagen II) and senescence-related molecules (p16 and p53), and activity of the p38 pathway were analyzed by Western blot assay. Briefly, total protein was extracted by RIPA lysis buffer (Beyotime, China) and its concentration was measured by the Protein BCA Kit (Beyotime, China). Then, 60 μg protein in each group was separated by SDS/polyacrylamide gel (SDS/PAGE) and transferred to the PVDF membrane (Beyotime, China). Subsequently, the PVDF membranes were incubated with primary antibodies (β-actin: Abcam, ab8227; p16: Novus, NBP2-37740; p53: Abcam, ab26; aggrecan: Norvus, NB120-11579; collagen II: Abcam, ab185430; p38: Cell Signaling Technology, #8690; p-p38: Cell Signaling Technology, #4511) and horseradish peroxidase (HRP)–conjugated secondary antibodies (Beyotime, China). Finally, the protein bands on the PVDF membrane were visualized by SuperSignal West Pico Trial Kit (Thermo, U.S.A.). The density of each protein band was quantitated using Quantity One Software.

After culture, AF cells were lysed using the RIPA lysis buffer (Beyotime, China), and the total protein was extracted according to the manufacturer's instructions. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the PVDF membrane. Then, PVDF membranes were sequentially incubated with primary antibodies (GAPDH: Abcam, ab181602; p16: Novus, NBP2-37740; p53: Abcam, ab26; NF-κB: Abcam, ab16502; phospho-NF-κB: Abcam, ab86299; all diluted at 1 : 2000) and secondary antibodies according to the standard procedure. After protein bands were developed using ECL Plus (Amersham Pharmacia Biotech, Umea, Sweden), densitometry analysis was finished using the ImageJ software (National Institutes of Health, USA).

Briefly, collected AF cells were lysed on ice in RIPA buffer (Beyotime, China) for 15 minutes and then centrifuged at 15,000 × g for 15 minutes at 4°C to collect cell lysates. After measuring protein concentration using a BCA Protein Assay Kit (Beyotime, China), equal protein samples in each group were resolved in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). After the PVDF membranes were incubated with primary antibodies (GAPDH: Abcam, ab8245; p16: Novus, NBP2-37740; p53: Abcam, ab26) overnight, they were incubated with secondary antibodies for 2 hours at room temperature. Finally, protein bands were visualized using the SuperSignal West Pico Trial Kit (Thermo, USA). Densitometric analysis of the protein bands was finished using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).