Expression of rPGRMC1 results in dexamethasone binding activity. Western blot for rPGRMC1 in various cell fractions using the anti-IZ Ab in COS-7 cells transfected with the indicated construct. All lanes were loaded with 10 μg protein/lane. Note, HC5 is a truncated form of rPGRMC1 cloned from rat kidney  (a). Western blot for rPGRMC1 using the anti-IZ Ab and CYP2E1 (lower blot). Rat hepatocytes were cultured for 24 hours to allow attachment (T0) and then treated for 24 hours with the indicated ligand or ethanol vehicle prior to analysis. Each lane contains 10 μg total protein/well, typical of 3 separate experiments (b). Confocal microscopy of rat hepatocytes demonstrating non-nuclear location of PGRMC1 and CYP2E1 (c). 200 × 106 COS-7 cells were transfected with pSG5-rPGRMC1, pSG5 or pcDNA3.1e/lacZ and 13,000 g cell extracts prepared and incubated with radiolabelled dexamethasone as outlined in methods section. Supernatant dpm after charcoal dextran treatment to remove free radioligand is given in dpm after normalisation of protein for total (specific and non-specific) – white bars; and non specific (by co-incubation of 1000-fold molar excess unlabelled dexamethasone) – black bars. The percentage of cells that stained positive for beta galactosidase activity (grey bars) was determined in situ in separate wells by examining at least 5 randomly selected low power fields. Data are the mean and standard deviation of at least 3 separate determinations from the same experiment, typical of 2 separate experiments (d). 200 × 106 COS-7 cells were transfected with pSG5-rPGRMC1. Dexamethasone binding activity was determined in whole COS-7 cells as outlined in methods section and in the presence of the indicated concentration of unlabelled potential competitor. Specific binding was determined by co-incubation of replicates also containing unlabelled 1000-fold molar excess of unlabelled dexamethasone. Typically, non specific binding accounted for between 40–60% of total binding of radioligand. Data are the mean and standard deviation of 3 separate determinations from the same experiment, typical of 3 separate experiments. Control is the mean and standard deviation specific activity of 3 determinations from the same experiment after subtraction of non-specific binding. The percent of binding in the presence of unlabelled competitors was determined after subtraction of non-specific binding. Data are typical of at least 2 separate experiments (e).