Anti hif 1α gtx127309

Cells were harvested and lysed in M-PER reagent (Thermo Fisher) containing cOmplete^™ protease inhibitor cocktail (Roche, Basel, Switzerland) and PMSF (Sigma). Each 10 to 15 µg of denatured protein samples were separated on Mini-PROTEAN^® TGX^™ Precast Gels (Bio-Rad) and transferred to methanol-activated Hybond P membranes (GE Healthcare, Pittsburgh, PA, USA). Membranes were blocked with TBS-T buffer containing 5% skimmed milk and incubated with anti-α-tubulin (sc-23948, Santa Cruz, Dallas, TX, USA), anti-DNMT3A (3598, Cell Signaling, Danvers, MA, USA), anti-HIF-1α (GTX127309, GeneTex, Irvine, CA, USA) or anti-HIF-2α (7096, Cell Signaling) antibodies. For detection of protein expression with multiple antibodies, membranes were cut into up to 3 pieces with a sufficient margins. Bands from multiple blots in a single experiment were normalized by Ponceau-S staining (Sigma-Aldrich) Supplementary Figure 3A–3F.

Cells were harvested in M-PER reagent (Thermo Fisher) containing complete™ protease inhibitor, PhoSTOP phosphatase inhibitor (Roche, Basel, Switzerland) and PMSF (Sigma). Each 10 μL of denatured samples were separated on Mini-PROTEAN^® TGX™ Precast Gels (Bio-Rad, Hercules, CA, USA) and transferred to methanol-activated Hybond P membrane (GE Healthcare, Pittsburgh, PA, USA). Membranes were blocked with TBS-T buffer containing 5% skimmed milk followed by incubation with anti-GLUL (GS) (HPA007316, Sigma), anti-β-actin (A2228, Sigma), anti-α-tubulin (sc-23948, Santa Cruz, Dallas, TX), anti-HIF-1α (GTX127309, GeneTex, Irvine, CA), anti-HIF-2α (7096, Cell Signaling, Danvers, MA, USA), anti-hydroxy-HIF-1α (Pro564, OH-HIF) (3434P, Cell Signaling), anti-GLUT1 (ab15309, Abcam, Cambridge, UK) or anti-VHL (2738, Cell Signaling) antibodies. For multiple protein detection, a membrane was cut into two or three pieces with a good margin. The uncropped images of immunoblots were shown in Supplementary Figures 5 and 6. Bands from multiple blots in a single experiment were normalized by Ponceau-S staining.