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).