Background subtraction and blot-to-blot adjustment of immunoblot data
Individual AGM samples were assessed on multiple immunoblots.For all signals (CYP2A, β-actin, and Coomassie blue-stain), software automatic background subtraction was applied to all blots. CYP2A signals were then normalized to β-actin signals or Coomassie blue-stained total protein to adjust for variation in protein loading (see Supplementary Tables 1 and 2). Further background subtraction was applied, after this normalization to loading controls, based on the y-intercept from the linear regression analysis of the CYP2A/ β-actin or CYP2A/Coomassie control group pooled standard curves present on each immunoblot. To reduce blot-to-blot variation in the analyses, the individual AGM signals were adjusted using the 75 μg of pooled Control group lung membrane signal (internal standard). Analyses with (illustrated in the figures), and without, this additional blot-to-blot adjustment had no impact on the fold or statistical significance of differences (Supplementary Tables 1 and 2).