Whole cell lysates from cells (human embryonic kidney cells-HEK 293) overexpressing CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1 respectively were used as positive controls for immunoblotting while lysates from cells containing vector only were used as negative controls. The cell lysates and their corresponding controls were obtained from (Novus Biologicals, Cambridge, UK). Cell lysates (5 μg protein/lane) were resolved by electrophoresis on NuPAGE 4-12% Bis-Tris gels (Fisher Scientific, Loughborough, UK). Following protein transfer to nitrocellulose membrane the membranes were washed for 45 minutes at room temperature in phosphate buffered saline-Tween-20 (PBST) containing 3 % (w/v) skimmed milk powder to block non-specific protein binding. Membranes were incubated overnight at 4°C with individual monoclonal antibodies diluted in PBST (1/2 dilution) and then washed 6 times for a total of 60 minutes in 1% skimmed milk. The membranes were subsequently probed for 60 minutes with a secondary antibody conjugated horseradish-peroxidase-conjugated anti-mouse IgG (1/2000, Sigma-Aldrich, Dorset, UK). Membranes were then washed (6 times) for a total of 60 minutes in 1% skimmed milk and protein bands visualized using the enhanced chemiluminescence detection system (Fisher Scientific) [26 (link), 49 (link)].
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