Pre stained protein standard

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).

After treatment with FA-1 (150 nM) and exposure to CSE, we harvested cell lysate with 1X RIPA buffer (Sigma-Aldrich), including protease and phosphatase inhibitor (Thermo Fisher). Protein concentration was determined with BCA protein assay reagent (Thermo Fisher). Protein samples were separated with SDS-PAGE and transferred to polyvinylidene fluoride membrane. The membrane was blocked with 5% non-fat milk in TBS with 0.5% Tween-20, and incubated with primary and second antibodies, and visualized with enhanced chemiluminescence reagent (Advansta). The approximate positions (kDa) of prestained protein standard (Bio-Rad) are indicated on the right of the blots. Equivalent loading was verified by stripping the blot and reprobing with antibodies to β-actin. Relative quantification of signal intensity was determined using image Lab software (Bio-Rad, Hercules, CA, USA) and expressed as a relative densitometry.