Fig. 2

Effect of VCE-004.8 on PDH activity and HIF-1α hydroxylation and stabilization. a MO3.13 cells were treated with VCE-004.8 for 6 h in the presence of MG132 and the levels of hydroxylated HIF-1α and HIF-1α were determined by immunoblot (n = 3). b HEK-293T cells were transfected with HA-PHD1, HA-PHD2, or HA-PHD3 as indicated. After 24 h of transfection, cells were treated as follows: P1, non-transfected cells; P2, cells were transfected with PHDs and immunoprecipitated with IgG-HA; P3, cells were transfected with PHDs and immunoprecipitated with αHA; P4, cells transfected with PHDs, stimulated with VCE-004.8 (2.5 μM) and immunoprecipitated with αHA; P5, cells transfected with PHDs, stimulated with CBD (2.5 μM) and immunoprecipitated with αHA; P6, cells transfected with PHDs, stimulated with DMOG (1 mM) and immunoprecipitated with αHA. HIF prolyl hydroxylase activity was measured using GST-HIF-1α protein, and the levels of hydroxylated HIF-1α, HIF-1α, and PHDs were analyzed by immunoblot (n = 3)