Figure 4A, The indicated cells transiently transfected with pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) were cultured under the indicated oxygen conditions for 24 hours and subjected to Western blotting using the indicated antibodies.The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.The indicated cells transiently transfected with pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated (B,D,E,F) The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.H, After transient transfection with the expression vectors for ZBTB2-V5, ZBTB2-myc, or their empty vector (-), ZBTB2-myc protein was immunoprecipitated using the anti-myc antibody and co-precipitated ZBTB2-V5 was detected using the anti-V5 antibody (upper). One-tenth of the whole cell lysate (WCL) was subjected to immunoblotting with the indicated antibodies (lower).I,J, The indicated cells transiently transfected with or without either pcDNA4/ZBTB2-LgBiT or pcDNA4/LgBiT and either pcDNA4/ZBTB2-SmBiT or pcDNA4/SmBiT, as indicated, were cultured under < 0.1% oxygen (I) or the indicated oxygen conditions (J) and subjected to the split luciferase complementation assay.K, HeLa cells overexpressing wildtype or the indicated mutant of ZBTB2 (ZF2 or ZF3-deletion) were cultured under < 0.1% oxygen conditions, and subjected to immunoprecipitation with anti-myc antibody. Coprecipitated DNA was subjected to the qRT-PCR experiment using primers against HRE regions of the STC1 promoter.L, The same experiment as I was conducted using the indicated combination of pcDNA4/ZBTB2-LgBiT (WT), pcDNA4/ZBTB2ΔZF2-LgBiT (ΔZF2), or pcDNA4/ZBTB2ΔZF3-LgBiT (ΔZF3), and pcDNA4/ZBTB2-SmBiT (WT), pcDNA4/ZBTB2ΔZF2-SmBiT (ΔZF2), or pcDNA4/ZBTB2ΔZF3-SmBiT (ΔZF3).