A subset of lambs (n = 4/treatment) was selected for analysis of cytochrome P450 subfamily 3A (CYP3A) activity using 30 µg microsomal protein from neonatal livers according to the procedures of Kumar et al. (2017) (link). Due to the ability to recognize several proteins, CYP3A subfamily was assessed rather than individual proteins (Acevedo et al., 2005 (link); Damiri et al., 2012 (link)). In brief, 10% polyacrylamide gel was used to separate proteins by gel electrophoresis (SDS-PAGE) and transferred to a 0.45 µm nitrocellulose (Bio-Rad). Molecular weight markers were a pre-stained protein standard (Bio-Rad). Wild-type mouse liver, 20 µg, was used as a positive control for antibody reactivity. The blot was blocked using 1% skim milk/0.1% Tween 20 suspended in phosphate buffered saline. Equal loading of samples was accomplished using rabbit anti-mouse β-actin (Sigma Aldrich, St. Louis, MO). The primary antibody was rabbit antirat CYP3A1 (Chemicon International, Temecula, CA) diluted 1:1000. Secondary antibody used for recognition of CYP3A subfamily was goat anti-rabbit IgG (Bio-Rad) alkaline-phosphatase diluted 1:500. A chemiluminescent kit (Bio-Rad) was used for visualization of the bands according to the manufacturer recommendations. Quantification of chemiluminescence was done using a Chemi-Doc system coupled with Image Lab software (Bio-Rad).
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