Western blotting

Cell Lysis Buffer for Western or IP (Beyotime Institute of Biotechnology) was used to extract the total protein. Protein concentration was detected using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). A total of 20–40 µg was loaded on an 8–12% SDS gel and resolved using SDS-PAGE. The resolved proteins were transferred to PVDF membranes (EMD Millipore) and were blocked in 5% non-fat milk (Yili Group) at room temperature for 1 h. Membranes were incubated with primary antibodies against HIF-1α (AF1009, Affinity Biosciences) and β-actin (sc-47778; Santa Cruz Biotechnology, Inc.) both at 1:1,000 at 4°C overnight. Incubation of membranes with horseradish peroxidase-conjugated goat anti-rabbit (A0208, 1:5,000; Beyotime) or anti-mouse (A0216, 1:5,000; Beyotime) secondary antibody was performed at 37°C for 45 min. An ECL reagent (Beyotime Institute of Biotechnology) was used to visualize the signals, and densitometry analysis was performed using Gel-Pro-Analyzer software (version 4; Media Cybernetics, Inc.).