Anti hif 1α

Fixed joint tissues embedded in paraffin were cut into 7-µm-thick sections, dewaxed using xylene, dehydrated through an alcohol series, and stained with hematoxylin and eosin, safranin O, or toluidine blue to detect proteoglycans. Endogenous peroxidase activity was quenched with methanol/3% H₂O₂. Immunohistochemistry was performed using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Sections were incubated with specific antibodies (i.e., anti-TNFα, anti-IL-1β, anti-IL-6, anti-IL-17, anti-RANKL, anti-VEGF, and anti-HIF-1α; all from R&D Systems) overnight at 4 °C. The tissue sections were then incubated with a biotinylated secondary antibody, followed by a streptavidin-peroxidase complex for 1 h. The final color product was developed using diaminobenzidine as the chromogen (DAKO, Carpinteria, CA, USA).

For IHC, paraffin-embedded kidney sections (3 µm) were deparaffinized, rehydrated, blocked, and incubated with various primary antibodies, including anti-CD68 (1:500, Boster Biological Technology, Wuhan, China), anti-Nephrin (1:100, Affinity Biosciences, Cincinnati, OH, USA), anti-TGF-β (1:500, Abcam, Cambridge, UK), and anti-HIF1α (5 μg/mL, R&D systems, Minneapolis, MN, USA) overnight at 4°C. Sections were then washed with PBS containing 0.1% Triton and incubated with HRP-conjugated secondary antibody (ZSGB Biotechnology, Beijing, China) for 1 h at room temperature. After the final wash with PBS/triton, sections were stained with DAB (ZSGB Biotechnology, Beijing, China) substrate and hematoxylin. The images of stained sections were acquired by bright field microscope, and quantitative analysis of positive staining areas (%) in images was done using ImageJ (National Institutes of Health, Bethesda, MD). Identical staining without the primary antibody was used as a negative control.

Lymphocytes were lysed in F buffer (1×10⁷ cells mL⁻¹) and subjected to immunoblot analysis as described previously [3] , [10] . Total ACC and ACC^S79 were detected by bi-fluorometric analysis using the Odyssey LICOR system and ImageJ software was used for integral signal quantification. Anti-AMPKα1 and anti-ACC^S79 were kindly provided by Grahame Hardie, University of Dundee, U.K. Anti-Smc1 was purchased from Bethyl Laboratories Inc and anti-Hif1α was obtained from R&D Systems. Anti-Glut1 was a gift from Geoff Holman, University of Bath, U.K. [19] , [24] (link). All other antibodies for immunoblotting were obtained from Cell Signaling Technology. Antigens were detected using suitable HRP-conjugated secondary antibodies and enhanced chemoluminescence.

Samples were solubilized in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride for 1 h at 4 °C. Cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride transfer membrane. The membranes were stained using the following primary antibodies: anti-HIF-1α, anti-HIF-2α (1:1000 dilution; both from R&D Systems, Minneapolis, MN, USA), anti-CXCR4 (1:1000 dilution; Abcam, Cambridge, UK), anti-Akt (1:1000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-Akt, anti-extracellular signal-regulated kinases (Erk) 1/2, anti-phospho-Erk1/2 (1:1000 dilution; all from Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (1:5000 dilution; Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were anti-rabbit IgG-horseradish peroxidase (HRP), anti-mouse IgG-HRP (1:3000 dilution; both from Cell Signaling Technology, Danvers, MA, USA), and anti-goat IgG-HRP (1:2000 dilution; R&D Systems, Minneapolis, MN, USA). The membranes were exposed to the ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA) using an enhanced chemiluminescence detection system. Signal intensity was analyzed with Quantity One software (Bio-Rad, Hercules, CA, USA).

PCL (M_n = 700,000–900,000), sodium orthovanadate, sodium fluoride, β-glycerophosphate, DOX (doxorubicin), and BrdU were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pimonidazole hydrochloride was obtained from Hypoxyprobe Inc. (Burlington, MA, USA). The following antibodies were used for the study: Alexa Fluor 594 phalloidin (Thermo Fisher Scientific, Waltham, MA, USA), anti-HIF-1α (R&D Systems, Minneapolis, MN, USA), anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA), anti-F-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-BrdU (Thermo Fisher Scientific, Waltham, MA, USA), anti-pimonidazole (Hypoxyprobe Inc.), anti-Ki67 (Cell Signaling Technology, Beverly, MA, USA), and cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA). The following secondary antibodies were used: Alexa Fluor 488-conjugated anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 594-conjugated anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 594-conjugated anti-rabbit (Thermo Fisher Scientific, Waltham, MA, USA), and HRP-conjugated anti-mouse (Cell Signaling Technology, Beverly, MA, USA).

Immunoblot analyses were carried out as previously described [15 (link)]. We used rabbit polyclonal anti-ALAD (Abcam, Cambridge, UK), mouse monoclonal anti-Actin (MP Biomedicals, Santa Ana, California, USA), mouse monoclonal anti-COX4I1 (Santa Cruz Biotechnology, Dallas, Texas, USA), goat polyclonal anti-HIF-1α (R&D systems, Minneapolis, USA), rabbit polyclonal anti-HSP90 (Santa Cruz Biotechnology, Dallas, Texas, USA), and mouse monoclonal anti-PPOX (Abcam, Cambridge, UK) as the primary antibodies, depending on the purpose of the immunoblot analysis. For the second antibody, we used horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody, HRP-conjugated anti-rabbit IgG antibody (Cell Signaling Technology, Beverly, Massachusetts, USA), and HRP-conjugated anti-goat IgG antibody (Santa Cruz Biotechnology, Dallas, Texas, USA) at 1:3000 dilution.