4.9. PHD2 Activity Assay

To analyze the in vitro activity of PHD2, we used biotinylated HIF-1α oxygen-dependent degradation domain (Biotin-DLDLEALAPYIPADDDFQL; from amino acids 556 to 575 of HIF-1α) and hydroxylated control (Biotin-DLDLEALAP[OH]YIPADDDFQL) peptides immobilized on streptavidin and bovine serum albumin (BSA)-coated 96 well plates. After exposure to hypoxia, cardiomyocytes were harvested and lysed with hypotonic buffer (20 mM HEPES; pH 7.4, 5 mM NaF, 10 μM Na₃VO₄, 0.1 mM EDTA, protease inhibitor cocktail, and 2 mM DTT) for 20 min. Then, NP-40 at a 0.5% final concentration was added. Equal quantities of protein (50 μg/well) were incubated with reaction buffer (Tris-Cl; pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 2 mM DTT, 0.5 mM 2-OG, and 1 mM ascorbate) for 1 h at RT. Samples were incubated with anti-hydroxy-HIF-1α primary antibody (3434P; Cell Signaling Technology), followed by the addition of goat anti-rabbit HRP-conjugated secondary antibody. Peptide hydroxylation was detected at 650 nm using TMB substrate.