Enhanced chemiluminescence

Tissue samples and cells were lysed in Protein Extraction Solution (RIPA or whole cell lysate buffer). We purchased RIPA buffer from Elpis-Biotech (South Korea). Whole cell lysate buffer: 10 mM HEPES (pH 7.9), 400 mM NaCl, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, and 1 mM PMSF with proteinase inhibitor cocktail (Thermo Fisher Scientific). Samples were heated with equal volumes of SDS buffer and 2-mercaptoethanol at 100°C for 5 min, and each sample was loaded onto Tris-glycine gel. After electrophoresis and transfer, PVDF membranes (Millipore) were blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% skim milk (Lab Scientific). The antibodies used in this study were as follows: HIF-1α (BD Biosciences); HO-1 (Enzo Life Biosciences); LKB1, COX2, CaMKKβ, ERRα, PGC-1α, SIRT1 (Santa Cruz Biotechnology); AMPKα, p-AMPKα (Thr¹⁷²) (Cell signaling technology); PHD2 (Novus Biologicals, Littleton, USA); β-Actin (Sigma). Membranes were incubated with peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence (Elpis-Biotech). Bands from western blotting (S1 File) were analyzed using Image J.

Cells were lysed in radioimmunoprecipitation assay buffer (R4100; GenDEPOT, Katy, TX, USA) containing protease and phosphatase inhibitors (Sigma-Aldrich). The soluble fraction of cell lysates was isolated by centrifugation at 13,000 rpm for 30 min at 4 °C. The protein concentration was measured using a protein assay (Bio-Rad, Hercules, CA, USA) and 20–40 μg was resuspended in Laemmli buffer and incubated for 5 min at 95 °C before proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride membrane (Bio-Rad). After incubation in Tris-buffered saline containing 0.1% Tween 20 and 5% bovine serum albumin (BSA) for 1 h, the blot was incubated overnight at 4 °C with primary antibody. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h, and protein bands were visualized by enhanced chemiluminescence (ElpisBiotech, Daejeon, Korea). Uncropped images of blots are shown in Supplementary Fig. 15.