Cells were washed with ice-cold PBS, harvested by scraping, and then homogenized in lysis buffer (CelLytic M, Sigma-Aldrich) supplemented with phosphatase and proteinase inhibitors (PhosSTOP and cOmplete, Roche). Lysates were incubated (1 hour, 4°C) and then centrifuged at 14,000g (10 min, 4°C). Protein samples were quantified using the Pierce BCA Protein Assay (Thermo Fisher Scientific), resolved by SDS–polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with TBST [50 mM tris, 150 mM NaCl, and 0.5% Tween 20 (pH 7.5)] containing 5% skim milk powder and probed overnight with primary antibody against SDHD (Millipore), p-STAT and STAT1 (Cell Signaling Technology), GFP (Abcam), or HIF1a (BD Biosciences). Bound antibodies were detected by horseradish peroxidase–linked secondary antibodies and processed with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).

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