Nb100 303

Western blotting and immunoprecipitation were performed using previous methods.^{60 (link)} HIF-1α antibody (1:1 000, 10006421; Cayman), VHL antibody (1:1 000, sc-5575; Santa Cruz), PHD1 antibody (1:1 000, NB100-310; Novus), PHD2 antibody (1:1 000, NB100-2219; Novus), PHD3 antibody (1:1 000, NB100-303; Novus), HSP70 antibody (1:500, ADI-SPA-812; Enzolife), HSP90 antibody (1:500, sc-13119; Santa Cruz) and β-actin antibody (1:2 000, A2066; Sigma) were used for immunoblotting. Protein bands were quantified using ImageJ. For immunoprecipitation, nuclear extracts from cultured B cells were incubated with Dynabeads protein G and 5 μg anti-HIF-1α antibody (H1a67) overnight at 4 °C. Anti-HIF-1α, (1:1 000, 10006421; Cayman) anti-HSP70 (1:500, ADI-SPA-812; Enzolife) and anti-ubiquitin (1:500, sc-166553; Santa Cruz) antibodies were used for immunoblotting. Western blot source data are included in Fig. S9.

Cells were lysed with 300 µL 1X Lysis buffer (Cell Signaling Technologies, Inc., Danvers, MA, USA) with 1 μg·mL⁻¹ 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) protease inhibitor (Sigma-Aldrich, Inc.). Total cell lysate protein concentrations were determined with the Better Bradford Kit (Thermo-Fisher Scientific) according to the manufacturer’s instructions. Western blot analysis was performed with 50 µg of MPC2 or 40 µg of U2OS cellular lysates. The blots were incubated with primary antibody to hypoxia-inducible factor 1-alpha (HIF1α) (NB100–449; Novus Biologicals, Littleton, CO, USA) Phd1 (Abcam; ab108980), Phd2 (Novus; NB100-2219), Phd3 (Novus: NB100-303) overnight, then incubated with secondary antibody at 1:2 000 (anti-rabbit–horseradish peroxidase [HRP]; Cell Signaling Technologies). Blots were stripped using SDS-glycine and reprobed with 1:15 000 anti-β-actin-HRP (A3854; Sigma-Aldrich). Detection was performed using the ECL Prime Western Blotting Detection Reagents (Amersham-GE Healthcare, Pittsburgh, PA, USA) and the GE AB1600 digital imager.