Figure 2.

Determination of complex formation between CYP1A2 and HO-1—Effect of POR. A, HEK 293T/17 cells were transfected with plasmids coding for rabbit CYP1A2-GFP, Rluc-HO-1, in the absence and presence of untagged-POR. The CYP1A2-GFP–Rluc-HO-1 BRET pair was measured 24 h after transfection in the absence (blue) and presence (red) of 500 ng of cotransfected POR DNA. Error bars represent the standard deviation (SD) of triplicate measurements of cells from a single transfection and generally do not exceed the size of the points. The experiment was performed three times with small adjustments to transfection conditions for optimization of protein expression levels. Results from each transfection were consistent. B, total protein expression was estimated from the sum of the fluorescence of the GFP and luminescence of the Rluc tags, using a GFP–Rluc fusion protein as a standard. C, effect of changes in protein concentration at a fixed ratio of GFP-CYP1A2 to Rluc-HO-1. HEK 293T/17 cells were transfected with different amounts of DNA, while maintaining an excess of the CYP1A2-GFP–tagged protein. The relative levels of GFP–Rluc protein expression were in excess of 38:1. BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; HO-1, heme oxygenase 1; POR, NADPH-cytochrome P450 reductase.