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Fig. 6. CBP enhances colocalization between CFP-HIF-1
and YFP-Arnt. (A) Cells were transfected with 400 ng of pCFP-mHIF-1 (CFP-HIF-1), pYFP-Arnt (YFP-Arnt) and pRc/RSV-CBP-HA (CBP) or pSG5/hSRC-1 (SRC-1) as indicated and analyzed as described in the legend of Fig. 1A. 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. (B) Colocalization between CFP-HIF-1 and YFP-Arnt. HEK 293 cells were transfected with 300 ng pYFP-Arnt (YFP-Arnt) and 700 ng pCFP-mHIF-1 (CFP-HIF-1). 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. (C) Protein expression levels of CFP-HIF-1 and YFP-Arnt under the conditions described in B. 36 hours after transfection, medium was changed and cells were given a further 2 hours at normoxia or in the presence of 100 µM CoCl2. 50 µg whole cell extracts were submitted to SDS-PAGE followed by immunoblotting using anti-GFP (-GFP) antibodies. (D) The CFP-HIF-1/YFP-Arnt heterodimer efficiently activates transcription of an HRE-driven reporter gene in a hypoxia-inducible way. HEK 293 cells were transfected with 500 ng HRE-driven reporter gene, 50 ng of the indicated expression plasmid and carrier DNA, pFlag-CMV-2, to keep the total DNA concentration at 1 µg. 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 with transfection of the pECFP-C1 and incubated at normoxia. Relative luciferase values obtained with expression of CFP-HIF-1 at normoxia (^) or hypoxia (*) in the presence or absence of expressed YFP-Arnt are significantly different (P<0.05). Values represent the mean ± s.e.m. of three independent experiments performed in duplicate.
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