Figure 3.

Effect of HO-1 on the interaction between CYP1A2-GFP and POR-Rluc.A, HEK 293T/17 cells were transfected with plasmids coding for rabbit CYP1A2-GFP and POR-Rluc in the absence and presence of 500 ng of unlabeled HO-1. Twenty-four hours post transfection, cells were collected and BRET was measured. When cells were cotransfected with HO-1 DNA, the maximum BRET signal generated was significantly lower (red) than that of cells transfected with the CYP1A2-GFP•POR-Rluc pair alone (blue). Data points represent triplicate measurements of cells from a single transfection; error bars represent the standard deviation (SD) and generally did not exceed the size of the points. Each experiment was repeated with small adjustments to transfection conditions for optimization of protein expression. Results were both transfections were consistent. B, total protein expression was estimated from the sum of the fluorescence of the GFP and luminescence of the Rluc tags. BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; HO-1, heme oxygenase 1; POR, NADPH-cytochrome P450 reductase.