Anti calpain 2

Total proteins were extracted by RIPA buffer (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime, Shanghai, China). The protein concentration was measured by the BCA Protein Assay Kit (Thermo Fisher, Waltham, USA). Then, equal amounts of protein were loaded on 8–12% SDS-PAGE gels and transferred onto a PVDF transfer membrane (Millipore, Massachusetts, USA). After blocking with 5% skimmed milk solution, the membranes were incubated with specific primary antibodies overnight at 4°C followed by further incubation with the secondary antibody at room temperature for 2 h. Both primary and secondary antibodies were purchased from Thermo Fisher (Waltham, USA), including anti-β-actin, anti-Nrf2, anti-HO-1, anti-p-NF-κB, anti-IL-1β, anti-TNF-α, anti-Bip, anti-calpain-2, anti-active caspase-12, anti-active caspase-3, anti-Bax, anti-Bcl-2, anti-ACAN, anti-Col-II, anti-Col-I, anti-MMP-13, anti-aggrecanase 1, and secondary antibody IgG. Finally, the protein bands were detected using the ECL Plus Reagent (Thermo Fisher, Waltham, USA), and their densitometry was analyzed with the ImageJ software version 8.0(Bio-Rad, Hercules, USA). With β-actin as control, the expression of total protein was normalized.

Thirty μg proteins were subjected to SDS-PAGE, and the proteins on gels were transferred to PVDF membranes. The membranes were blocked with 5% skim milk, and incubated with anti-AR (1:1000, Cell Signaling Technologies, Inc., Danvers, MA, USA), anti-calpain-1 (1:1000, Thermo Fisher Scientific, Waltham, MA, USA), anti-calpain-2 (1:1000, Thermo Fisher Scientific, Waltham, MA, USA), anti-calpastatin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) or GAPDH (1:1000, KeyGEN Biotech, Nangjing, China), then with a horseradish peroxidase-conjugated secondary antibody (1:5000, ZSGB-BIO, Beijing, China). The proteins were visualized using an ECL Western blot kit (Beyotime Biotechnology, Haimen, Jiangsu, China) and document with FluoChem E imaging systems (ProteinSmiple, San Jose, CA, USA).