Ab184714

Tissues or cells were lysed on ice in 1× RIPA lysis and extraction buffer (cat. #89900, Thermo Fisher Scientific) with Halt protease inhibitor (cat. #78441, Thermo Fisher Scientific). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (cat. #23225, Thermo Fisher Scientific). Total protein (20 µg) was loaded onto the gels, and blots were developed with primary antibodies against PHD3 (ab184714, Abcam, Cambridge, UK; 1:2000 dilution); HIF1α (D2U3T, Cell Signaling Technology, Danvers, MA, USA; 1:1000 dilution); HIF2α (D9E3, Cell Signaling Technology; 1:1000 dilution); fumarate hydratase (ab95950, Abcam; 1:3000 dilution); EGFR (sc-373746, Santa Cruz Biotechnology, Dallas, TX; 1:1000 dilution). pEGFR (sc-57545, Santa Cruz Biotechnology; 1:200 dilution); glutaminase (GLS1; ab93434, Abcam; 1:1000 dilution), glutaminase 2 (GLS2; NBPI-76544, Novus Biologicals, Littleton, CO, USA; 1:1000 dilution); alanine-serine-cysteine transporter 2 (ASCT2; ABN73, Millipore Sigma, 1:1000 dilution), and β-actin-horseradish peroxidase (5125S or 12262S, Cell Signaling Technology; 1:5000 dilution). Image J (National Institutes of Health, Bethesda, MD, USA) or Image Studio Lite (version 5.2, LI-COR Biosciences, Lincoln, NE, USA) were used to perform densitometry.