Relationship between ING4 and HPH-2 in JJN3. JJN3 was treated with or without hypoxic condition for 12 hours and then nuclear extracts were evaluated for HPH-2 expression by Western blot analysis. Histone H1 was used as internal control (A). JJN3 was transfected by electroporation with 1 nmol smart pool double-stranded RNA oligonucleotides (siRNA) against ING4 or HPH-2 or with a nonspecific control siRNA (Cy). After 24 hours, cells were incubated in the presence or absence of hypoxic condition for 12 hours. ING4 and HPH-2 mRNA expression was quantified by real-time PCR. Graphs represent the mean plus or minus SD of −ΔCt value (B). Nuclear lysates of JJN3 overexpressing ING4 were immunoprecipitated with anti–HPH-2 Ab or anti-IgG control and with anti-ING4 Ab or anti-IgG control. ING4 coimmunoprecipitated with HPH-2 and HPH-2 coimmunoprecipitated with ING4 were detected by Western blot analysis using anti-ING4 and anti–HPH-2 Abs, respectively (C). JJN3 was pretreated with the HPH-2 inhibitor DMOG (0.2 mM) for 2 hours or transfected by siRNA against HPH-2 or with a nonspecific control siRNA (Cy) and then incubated with or without hypoxic condition for 12 hours. HIF-1α protein level was detected in nuclear lysates by Western blot analysis (D left). HIF-1α activity was quantified by a transcriptional factor assay kit as described in “NF-κB and HIF-1α activity.” Nuclear extracts of COS-7 treated with CoCl₂ in the presence or absence of wild-type (wt) or mutated (mt) competitor oligonucleotides were tested as control (C). Graphs represent the mean plus or minus SD of HIF-1α activity normalized for 10 μg protein analyzed of 2 independent experiments performed in triplicate (OD = optical density; *P = .01; **P = .001; D right).