Figure 5. Decreased enzymatic activity of mutant R117W and G329V CYP1B1 proteins

A: Detection of the protein expression levels attained by the HA-CYP1B1 recombinant constructs in transiently transfected HEK293T cells (two separate replicates). pcDNA3.16 and pcDNA3.17 control cells transfected with the empty vector; mut4791-cells transfected with the G329V CYP1B1 construct; mut4154-cells transfected with the R117W CYP1B1 construct; CYP1B1 wt-cells transfected with the wild-type CYP1B1 cDNA. B: CYP1B1 activity in transiently transfected HEK293T cells expressed measured by a modified luciferase assay and expressed as arbitrary units compared to the positive wild-type CYP1B1 cDNA, which is considered 100%. The final values have been obtained after normalization against total cell protein, endogenous cellular CYP1B1 activity and transfection efficiency per construct and well. The standard deviation (5 replicates in two different experiments) is also indicated by bars. The decrease in activity for each mutant, down to 60% for the R117W and to 30% for the G329V, is statistically significant (Mann-Whitney test, p<0.01) and it is indicated with an asterisk.