Expression and regulation of HIF-1α and HIF-2α. (A) HIF-1α and HIF-2α mRNA were expressed in REPCs. HIF-1α mRNA was constitutively expressed, whereas HIF-2α was significantly up-regulated by hypoxia (1% O2 for 6 hours). Shown are the means ± SD; n = 6; *P < .05. (B) Accumulation of HIF proteins during hypoxic incubation. HIF-1α protein accumulation peaked after 6 hours of hypoxic incubation. The HIF-2α protein became detectable after 1 hour of hypoxic incubation and remained elevated for up to 24 hours. Shown are representative immunoblots for HIF-1α and HIF-2α. α-Tubulin served as the loading control. Normoxic controls have been separated to indicate a repositioned lane from the same gel. (C) ChIP analysis of HIF-1 and HIF-2 DNA binding to the HRE of the EPO enhancer. Maximal HIF-1 binding was detected after 6 hours of hypoxia; after 24 hours, no HIF-1 binding to the EPO enhancer was observed. HIF-2 binding was detectable even after 24 hours under hypoxic conditions. (D) Regulation of the HIF-modifying prolyl hydroxylases and HIF target genes PHD2 and PHD3. Expression of both PHDs was continuously up-regulated by hypoxia. PHD mRNA expression was measured by real-time PCR and normalized to β-actin. PHD2 and PHD3 proteins were detected by immunoblot after 6 hours of hypoxic incubation. Shown are the means ± SD; n = 6; **P < .01.
