β actin

The soluble protein was prepared in RIPA lysis buffer (Beyotime, China) added with 1% phenylmethanesulfonyl fluoride (PMSF) (Beyotime, China). Equivalent protein (20–50 μg) was subjected to SDS-PAGE and transferred to electrophoretically PVDF membranes. After being immersed in blocking buffer (EpiZyme, China) at room temperature for 10 min, the membranes were incubated with specific primary rabbit antibodies at 4°C overnight. This is followed by incubating with secondary antibodies (1:5000, Beyotime, China) at room temperature for 1 h. Then, the antigen–antibody complex was detected by enhanced chemiluminescence (ECL) reagent. GAPDH and β-actin were used as loading controls.28 (link) The primary antibodies were used as follows: GAPDH (1:5000, Abcam, UK), β-actin (1:5000, Abcam, UK), Bcl-2 (1:2000, Abcam, UK), Bax (1:3000, Abcam, UK), Beclin-1 (1:1000, CST, USA), LC3 (1:1000, CST, USA), P62 (1:1000, CST, USA), AMPK (1:1000, Waneibio, China), phospho-AMPK (1:1000, Waneibio, China), mTor (1:500, Waneibio, China), phospho-mTor (1:500, Waneibio, China).

Total proteins were extracted from cells or mice tissues with RIPA buffer (CST, USA). Protein concentrations were measured using the BCA Protein Quantification Kit (Abbkine, USA). The same amount of protein samples were divided by SDS-PAGE (12% polyacrylamide gels), followed by transferring to PVDF membranes (Millipore, USA). The membranes were blocked with 5% milk and incubated with the primary antibodies for WTAP (1:1000) (Abcam, USA), Tsg101 (1:1000) (Abcam, USA), CD63 (1:1000) (Abcam, USA), Grp94 (1:1000) (Abcam, USA), caspase3 (1:1000) (Abcam, USA), PARP (1:1000) (Abcam, USA), and β-actin (1:1000) (Abcam, USA) at 4 °C overnight, in which β-actin served as the control. Then, the corresponding second antibodies (1:1000) (Abcam, USA) were used for hatching the membranes at room temperature for 1 h, followed by visualization using an Odyssey CLx Infrared Imaging System.

Chondrocyte pellets were stored at −80 °C until assayed. Frozen samples were pulverized under liquid nitrogen and placed in a homogenization buffer (10 mM phosphate buffer, 250 mM sucrose, 1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride or phenylmethylsulfonyl fluoride, and 0.1% [v/v] Tergitol, pH 7.5). Homogenates were centrifuged at 27,000g for 10 min at 4 °C to harvest supernatant for immunoblotting with antibodies. Immunoblotting was performed using primary monoclonal antibodies against PPAR-δ (1:2,000 dilution; Abcam, Cambridge, MA, USA), type II collagen (1:1,000 dilution; EMD Millipore, Billerica, MA, USA), TGF-β (1:1,000 μg/mL; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and β-actin (1:10,000 dilution; Abcam) and secondary antibodies tagged horseradish peroxidase (HRP) (rabbit for PPAR-δ and TGF-β, 1:20,000 dilution, mouse for type II collagen and β-actin, 1:5,000 dilution). Cell pellets were processed for NuPAGE gel (Invitrogen). Proteins separated in the gel were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using TE77XP semidry blotter with 10 V for 3 h (Hoefer, Inc., Holliston, MA, USA). Protein band signals on blots were detected on Amersham Hyperfilm enhanced chemiluminescence (GE Healthcare Life Sciences) using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Rockford, IL, USA).