Superoxide dismutase 1

The proteins were isolated from MEFs as described previously [87 (link)]. Proteins at a concentration of 20 μg/μL were separated by SDS-PAGE and subsequently transferred onto a PVDF membrane (Roche, Basel, Switzerland). After blocking, the membranes were incubated overnight at 4 °C with the following primary antibodies: Hif-1α, Cell Signaling Technology D2U3T #14179; catalase, Abcam ab16731; superoxide-dismutase 1, Abcam ab16831; fatty acid synthase, Santa Cruz sc-48357; trifunctional enzyme subunit beta, mitochondrial, Hadhb, Santa Cruz sc-271496; phospho-AMPK (Thr172), phosphorylated AMP-induced protein kinase, Cell Signaling (40H9). An appropriate horseradish peroxidase (HRP)-conjugated secondary antibody was utilized for chemiluminescence detection. Total protein normalization was achieved using AmidoBlack (Sigma Aldrich, St. Louis, MO, USA). Immunoblots were detected using the Alliance 4.7 Imaging System (UVITEC, Cambridge, UK) and an enhanced chemiluminescence kit (Thermo Fischer Scientific, Waltham, MA, USA). Experiments for each protein of interest were performed at least two times.

The protein level in the renal tubule cells and kidney tissues was determined by Western blotting as previously described^{15 (link)}. The sodium dodecyl sulfate (SDS)-PAGE was used to separate equal amount of proteins, and then transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies for GRP78 (#3183), Chop (#2895), Bax (#2772), Bcl-2 (#15071), cleaved caspase-3 (#9664), Sirtuin-1 (Sirt1; #8469), phosphorylated NFκB-p65 (#3033) (Cell Signalling, Danvers, MA, USA), catalase (#ab16731), superoxide dismutase 1 (SOD1; #ab13498), Klotho (#ab203576), CD68 (#ab125212) (abcam, Cambridge, MA, USA), NFκB-p65 (#sc-8008), cyclooxygenase (Cox)-2 (#sc1745), β-actin (#sc-47778) (Santa Cruz, Dallas, TX, USA), inducible nitric oxide synthase (iNOS; #610329) (BD, Franklin Lakes, NJ, USA), and Ly6G (#14-5931-82) (eBioscience, San Diego, CA), and then incubated with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA). The signals were detected by using enhanced chemiluminescence substrates (Bio-Rad), and developed with a Fuji Blue X-Ray Film. Protein bands were quantitated by using ImageJ software. The raw data/full blot summary is available in the Supplementary Fig. 5.