Ab194583

To verify the purity of the ALR IgG, 20 µg ALR was processed for SDS-PAGE, and the bands were visualized by silver staining using the Fast Silver Stain Kit (Beyotime). For western blotting, proteins were extracted from cells using RIPA lysis buffer (Beyotime), and protein concentrations were determined with a bicinchoninic acid (BCA) Protein Assay (Beyotime). For western blotting, proteins (30 µg/well) were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, blocked with 5% nonfat milk, and probed with ALR (1:1,000), Bax,1:2,000 (ab182733; Abcam, Cambridge, UK), Bcl-2, 1:2,000 (ab194583; Abcam), Cleaved caspase-3 (1:1,000; Cell Signaling Technology), and cleaved caspase-9 (1:1,000; Cell Signaling Technology) primary antibodies.

Post 12 hours of CME modeling, rats were euthanized. Thick sections (4μm) were prepared, followed by immunofluorescence staining as suggested by the manufacturer. These sections were rinsed thricely in PBS, and then they were blocked in BSA for 0.5 hours at ~25 °C, followed by incubating the sections with following primary antibodies for 24 hours (at 4 °C): cleaved caspase-3 (#9661, Cell Signaling Technologies, Danvers, MA, USA), Bax (ab53154, Abcam, Cambridge, UK), and Bcl-2 (ab194583, Abcam) followed by five times washing with PBS. The sections were then subjected to incubation with fluorescent secondary antibodies at ~ 25°C for 50 minutes. DAPI was used for counterstaining of nuclei for 7 minutes. A fluorescence microscope (Olympus, Japan) was employed to obtain the images.

Immunohistochemistry was performed using the method described by Giribabu etal.27 (link) Kidney sections were deparaffinized, rehydrated, and antigen-retrieved using a10 mM sodium citrate buffer solution (pH 6.0) for immunohistochemical investigations. Endogenous peroxidase activity was suppressed with 0.3% Hydrogen peroxide (H₂O₂) prior to sections being incubated overnight at 4°C with primary antibodies such as Anti-Nrf2 (ab62352), anti-NOX-4 (ab109225), anti-Keap-1 (ab218815), anti-NF-κB (ab16502), anti-MCP-1 (ab25124), anti-BCL-2 (ab194583) and anti-BAX (ab32503), anti-Caspase-9 (ab184786) (1:1000; Abcam, Cambridge, UK) diluted in 5% bovine serum albumin (BSA). After incubation, slides were rinsed three times in PBS, incubated for 1hour with the appropriate secondary antibody for 60 min, stained with 3ʹ-Diaminobenzidine (DAB), and counterstained with hematoxylin. The images were examined and captured using an Olympus phase-contrast microscope.

Chondrocytes were lysed by radio immunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor. The concentration of proteins was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Beyotime Biotechnology, China). The proteins (30 µg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked in a nonfat dry milk solution for 1 hour. Then, the membranes were incubated overnight at 4°C with anti-AMPK (CST, #2532, 1:1000 dilution), anti-p-AMPK (CST, #2535, 1:1000 dilution), anti-mTOR (CST, #2983, 1:1000 dilution), anti-p-mTOR (CST, #5536, 1:1000 dilution), anti-Beclin-1 (Abcam, ab207612, 1:1000 dilution), anti-ULK1 (Abcam, ab133747, 1:1000 dilution), anti-LC3 II (Abcam, ab48394, 1:1000 dilution), anti-P62 (Abcam, ab56416, 1:2000 dilution), anti-Bcl2 (Abcam, ab194583, 1:2000 dilution) and anti-Bax (Abcam, ab32503, 1:1000 dilution) primary antibodies in dilution buffer. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Zhongshan Golden Bridge Biotechnology, 1:5000 dilution) at room temperature for 1 hour. Then, the membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA, USA).