Western Blot Analysis of Protein Targets

In detail, cells were collected and lysed in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholine, and 1 mM NaF), and the protein concentration was measured using a BCA protein assay kit (Pierce Biotechnology). Proteins were separated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After 2 h, the membranes were blocked with 5% skim milk and incubated with the primary antibodies at 4 °C overnight. After that, the membranes were washed with TBST for three times and incubated with HRP-labeled goat anti-rabbit antibody [Cell Signaling Technology (CST), USA] for 0.5 h. Finally, the immunoreactive signals were detected using the ECL detection system (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, IL, USA). The following primary antibodies were applied: rabbit anti-HIF1α (36169S, CST, USA), rabbit anti-P-p65 (3033S, CST, USA), rabbit anti-GPR116 (ab136262, Abcam, Shanghai, China), rabbit anti-p65 (8242S, CST, USA), rabbit anti-β-actin (4970 L, CST, USA), rabbit anti-GNA11 (ab153951, abcam, UK).