Fig. 2. CBP mediates in vivo interaction between HIF-1 and SRC-1. (A) Subcellular and intranuclear distribution of YFP-CBP, CFP-HIF-1 and YFP-SRC-1. HEK 293 cells were transfected with 400 ng of pYFP-CBP (YFP-CBP), pCFP-mHIF-1 (CFP-HIF-1) or pYFP-SRC-1 (YFP-SRC-1) and observed using fluorescence microscopy. The YFP-SRC-1-fluorescent signal is pseudocolored red. Bars, 3 µm. (B) SRC-1 colocalizes with CBP but not with HIF-1 in accumulation foci. Cells were transfected with 400 ng of expression plasmids for CFP-SRC-1 or YFP-SRC-1 and YFP-CBP or CFP-HIF-1, respectively. CFP- and YFP-SRC-1-fluorescent signals are pseudocolored red. Bars, 3 µm. (C) Redistribution of CFP-HIF-1 by YFP-SRC-1 is dependent on CBP. HEK 293 cells were transfected with 400 ng of pCFP-mHIF-1 (CFP-HIF-1), pYFP-SRC-1 (YFP-SRC-1) and pRc/RSV-CBP-HA (CBP). The bar chart shows the percentage of cells in which colocalization foci were observed at normoxia (N) or upon treatment with CoCl₂ (C). Bars, 3 µm. (D) YFP-SRC-1 enhances the hypoxia-dependent transactivation function of CFP-HIF-1. HEK 293 cells were transfected with 500 ng HRE-responsive reporter plasmid, 50 ng of the CFP or CFP-HIF-1 expression plasmids and 400 ng of YFP-SRC-1 expression plasmid. Total amount of DNA was kept at 1 µg using pFlag-CMV-2. After transfection, cells were kept at normoxia (21% O₂) or hypoxia (1% O₂) for 36 hours, harvested and luciferase activity was measured. Data are presented as fold induction over the luciferase activity obtained following transfection of cells using pECFP-C1 and incubation of the cells under normoxic conditions. Relative luciferase values obtained with expression of CFP-HIF-1 at normoxia (^) or hypoxia (^*) in the presence or absence of expressed YFP-SRC-1 are significantly different (P<0.05). Values represent the mean ± s.e.m. of three independent experiments performed in duplicate. (E) Expression levels of the different CFP- and YFP-fusion proteins. HEK 293 cells were transfected with 400 ng of each expression plasmid (as indicated). 36 hours after transfection medium was changed for treatment in the presence of absence of 100 µM CoCl₂ for 2 hours. 50 µg whole cell extracts were submitted to SDS-PAGE followed by immunoblotting using anti-GFP (-GFP) antibodies.