Oxygen-dependent hydroxylation of critical proline residues， catalyzed by prolyl hydroxylase (PHD1-3) enzymes， is a crucial posttranslational modification (PTM) within the canonical hypoxia-inducible factor (HIF)-centric cellular oxygen-sensing pathway. Alteration of substrates in this way often leads to proteasomal degradation mediated by the von Hippel-Lindau Tumor Suppressor protein (VHL) containing E3-ubiquitin ligase complex known as ECV (Elongins B/C， CUL2， VHL). Here， we outline in vitro protocols to demonstrate the ability of VHL to bind to a prolyl-hydroxylated substrate.
Oxygen-dependent hydroxylation of critical proline residues， catalyzed by prolyl hydroxylase (PHD1-3) enzymes， is a crucial posttranslational modification (PTM) within the canonical hypoxia-inducible factor (HIF)-centric cellular oxygen-sensing pathway. Alteration of substrates in this way often leads to proteasomal degradation mediated by the von Hippel-Lindau Tumor Suppressor protein (VHL) containing E3-ubiquitin ligase complex known as ECV (Elongins B/C， CUL2， VHL). Here， we outline in vitro protocols to demonstrate the ability of VHL to bind to a prolyl-hydroxylated substrate.
