Antibody specificity and protein linearity
Pooled Control group lung membranes from the lower and upper lung were serially diluted to generate a standard curve to assess the linear detection range of CYP2A, β-actin and total proteins (i.e. Coomassie blue-stained protein). Individual AGM samples were loaded at 75μg beside the pooled Control standard curve at 25μg, 75μg, and 150μg on each immunoblot. The standard curve was used to ensure that each membrane sample was in the linear range and to allow for blot-to-blot adjustments. Each blot had a pre-stained protein mixture in one lane for molecular weight estimation (PaperRuler Prestained Protein Ladder, ThermoFisher; Mississauga, ON, CA).
The specificity and sensitivity of the polyclonal anti-CYP2A6 antibody was assessed using positive controls including human liver microsomes, AGM liver microsomes, AGM lung membranes and membranes from cDNA expressed human CYP2A6 (Corning; Corning NY, USA), and CYP2A13 (XenoTech; Lenexa, KS, USA). Negative controls included cDNA expressed human CYP2E1 (BD Bioscience; Woburn, MA, USA), CYP2D6 (XenoTech; Lenexa, KS, USA), CYP2B6 (BD Bioscience; Woburn, MA, USA), and CYP3A4 (BD Bioscience; Woburn, MA, USA).