Af5001

Whole‐cell lysates of cell lines were prepared using radioimmunoprecipitation buffer (P0013J; Beyotime, Shanghai, China) according to the manufacturer's instructions. The protein concentration was determined by a bicinchoninic acid protein assay kit (23225; Thermo Fisher, Rockford, IL, USA). Subsequently, 40 μg of proteins was used for SDS/PAGE and transferred to poly(vinylidene difluoride) membranes. The membranes were blocked for 2 h in 5% nonfat dry milk in TBST and then incubated with the primary antibodies overnight at 4 °C. After incubation with the secondary HRP‐coupled antibodies for 2 h at room temperature, the membranes were washed with TBST, and the immunosignal was developed with enhanced chemiluminescence reagent and exposed in a ChemiDoc XRS + System (1708265; Bio‐Rad, Hercules, CA, USA). β‐Actin was used as the internal control. The concentrations and sources of primary antibodies are as follows: tissue inhibitor of metalloproteinase 1 (TIMP1; ab109125; Abcam, Cambridge, MA, USA) was 1 : 1000, plasminogen activator inhibitor 1 (PAI1; ab222754; Abcam) was 1 : 800, insulin‐like growth factor‐binding protein 1 (IGFBP1; ab181141; Abcam) was 1 : 1000, IL35 (LAC008Hu72; Cloud‐Clone Corp.) was 1 : 1000 and β‐actin (AF5001; Beyotime) was 1 : 2000.

Total cellular protein was extracted using RIPA lysis buffer containing protease inhibitor cocktail (Beyotime, China). The extracted proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, NY), which were subsequently blocked with a 5% solution of nonfat milk for 1 h. The membranes were then incubated with primary antibodies at 4°C overnight, followed by secondary antibody incubation for 1 h at room temperature. The proteins were visualized with a SuperLumia ECL HRP Substrate Kit (Millipore, NY) and detected using an LAS4000 chemiluminescence detection system (Fuji, Tokyo, Japan). The primary antibodies used were as follows: anti-β-actin (1:1,000, AF5001, Beyotime), SREBP1 (1:1,000, 14088-1-AP, Proteintech), and SCD1 (1:500, sc-515844, Santa Cruz Biotechnology). The anti-β-actin antibody was used as an internal control.

ARPE-19 cells were harvested and placed in cold radioimmunoprecipitation assay buffer (Beyotime) containing freshly prepared 2 mM PMSF and 1 X protease inhibitor cocktail (Beyotime). Aliquots of proteins (40 µg) were loaded into the lanes of a SDS polyacrylamide gel, and the proteins separated through electrophoresis were transferred onto nitrocellulose membranes. Subsequently, the membranes were blocked with 5% nonfat dry milk in 0.01 M PBS buffer (pH 7.4) and 0.05% Tween-20 for 1 h at room temperature. Afterward, the blocked membranes were incubated with appropriate primary antibodies against HIF-1α (1:2000), VEGFA (1:2000, Cat. No. ab46154, Abcam, Cambridge, MA, USA), and ANG1 (1:5000, Cat. No. ab95230, Abcam) overnight at 4 °C, and then with anti-rabbit or anti-mouse secondary horseradish peroxidase-conjugated antibodies (Amersham, Arlington Heights, OH, USA). GAPDH (1:5000, AF5001, Beyotime) was used as the loading control. The expression was determined using the enhanced chemiluminescence method (Amersham), and the density of immunoblots was measured with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).