Protease inhibitor cocktail

Proteins were extracted from iWAT using a radioimmunoprecipitation assay lysis buffer (Beyotime, catalog no. P0013B) containing protease inhibitor cocktail (1:100; Yeason, catalog no. 20104ES03). Following homogenization by homogenizer and lyse on ice, lysates were centrifuged at 12,000g for 15 min at 4°C. The concentration of each sample was calculated by the Bicin-choninic Acid method, and an equal amount of protein from each sample was added an equal amount of protein loading buffer. The protein extracts were denatured by metal bath at 100°C for 10 min. Equal amounts of protein extracts were separated by 10% SDS–polyacrylamide gel electrophoresis and were transferred to PVDF membranes. The membranes were blocked for 1 hour in 5% DifcoTM Skim Milk (BD Difco, catalog no. 232100), followed by immunoblotting with the following primary antibodies: anti–α-tubulin (mouse, 1:100; Sigma-Aldrich, catalog no. T6199) and anti-UCP1 (rabbit, 1:1000; Abcam, catalog no. ab10983) at 4°C overnight. Next day, the membrane was washed three times in Tris Buffered Saline with Tween for 3 × 5 min, followed by incubation with the following secondary antibodies: horseradish peroxidase (HRP)–goat anti mouse (1:5000; Earthox, catalog no. E030110) and HRP-goat anti rabbit (1:5000; Earthox, catalog no. E030120) for 1 hour at room temperature.

293T cells were collected and lysed in RIPA lysis buffer (Yeasen, Shanghai, China) supplemented with protease inhibitor Cocktail (Yeasen) for 30 min at 4 °C, centrifuged at 8800 rpm for 10 min, the supernatant was collected and divided into three 1.5 mL tube (one for input, one for IgG, and one for IP). For the immunoprecipitation assay, the cell lysate supernatants were incubated for 4 h at 4 °C with anti-Flag (Sigma) or anti-HA (Cell Signaling) coupled to the protein A/G-agarose beads (Sigma). Then, immunoprecipitated beads were washed 5–6 times with RIPA lysis buffer. Immunoprecipitates and input were subjected to run SDS-PAGE gel, and the next step was the same as the immunoblot assay.

Total protein from synovial tissue/cells was collected with RIPA lysis containing Phosphatase Inhibitor Cocktail and Protease Inhibitor Cocktail (Yeasen), then ground in a homogenizer. Concentrations of protein were determined by the BCA Protein Assay Reagent Kit. 10 μg protein was separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (Merck, NJ, USA). After blocking with 5% fresh nonfat milk in tris-buffered saline containing 0.05% Tween-20, the membranes were incubated with primary antibody overnight at 4 °C, followed by HRP-conjugated secondary antibody incubation. The membranes were incubated with Chemiluminescent HRP Substrate (Merck) for 1 min, then images were developed through Amersham Imager 600 (GE, MA, USA). The relative expression was quantified by ImageJ software.

Briefly, whole proteins were extracted with RIPA Buffer (#89900, Thermo Fisher, Waltham, USA) with protease inhibitor cocktail (#20124ES03, Yeasen, Shanghai, China). The extracts were separated by SDS-PAGE and then transferred to 0.45-μm immobilon-P transfer membrane (#IPVH00010, Millipore, Darmstadt, Germany). Membranes were blocked with 5% skim milk for 1 h at room temperature followed by incubation with a primary antibody at 4 °C overnight. Then the blots were detected with HRP conjugated secondary antibody and visualized with the Super ECL Detection Reagent ECL (#36208ES60, Yeasen).

Protein samples were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) with protease inhibitor cocktail and the protein concentration was measured using the BCA kit (Shanghai Yeasen Biotechnology). The Western blot was performed as previously described using vGLUT (1:1000, Synaptic System) and GAPDH (1:4000, Kangchen) as primary antibodies.

Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin-streptomycin solution, 0.25% trypsin/EDTA, and phosphate-buffered saline were purchased from Gibco. Fetal bovine serum (FBS) was purchased from Biological Industries. Rabbit monoclonal anti-Mfn1(Cat#: 14739S), rabbit monoclonal anti-Mfn2(Cat#: 9482S), rabbit monoclonal anti-Opa1(Cat#: 80471S), rabbit polyclonal anti-Drp1(Phospho Ser616 Cat#: 4494S), rabbit monoclonal anti-AMPK_α (Phospho Thr172 Cat#: 2535S), and rabbit monoclonal anti-β-actin(Cat#: 4970T) were purchased from Cell Signaling Technology. Rabbit monoclonal anti-Fis1(Cat#: ab156865) and rabbit monoclonal anti-DRP1(Cat#: ab184247) were purchased from Abcam. DMSO, Mito-Tracker, BCA Protein Quantification Kit, RIPA lysis buffer, protease inhibitor cocktail, phosphatase inhibitors, SDS-PAGE Gel Preparation Kit, and other Western blotting reagents were purchased from Yeasen (Shanghai, China). Annexin V-Alexa Fluor 647/PI Apoptosis Detection Kit was purchased from Fcmacs (Nanjing, China). The mitochondrial membrane potential assay kit with JC-1 was purchased from Beyotime (Nanjing, China). Unless otherwise indicated, all other chemicals and reagents were purchased from Sigma-Aldrich.