Microsomal or cytosolic proteins (30 µg in each lane) of hepatocytes were separated by SDS-PAGE and subsequently transferred onto nitrocellulose membranes (0.45 µm) using a
Trans-Blot ® Turbo™ Transfer System (Bio-Rad, USA). The membranes were blocked in 5 % non-fat dry milk/TBS-Tween-20 for 2 h. For the immunodetection of CYP1A1, CY-P1A2, GSTA and NQO1, the membranes were probed overnight with primary antibodies [CYP1A1 -rabbit polyclonal, 1:1000 (Novus Biologicals, USA), CYP1A2 -mouse monoclonal, 1:1000 (Novus Biologicals), GSTA -goat polyclonal, 1:3000 (Abcam, UK), NQO1 -rabbit monoclonal, 1:3000 (Novus Biologicals)] diluted in TBS-Tween 20 supplemented with 1 % BSA, washed four times with TBS-Tween 20 buffer and probed with complementary secondary antibodies for 1 h [
bovine anti-goat IgG-HRP, 1:3000, Santa Cruz Biotechnology (USA), bovine anti-mouse IgG-HRP, 1:10 000, Santa Cruz Biotechnology,
bovine anti-rabbit IgG-HRP, 1:10 000, Santa Cruz Biotechnology]. The signal was detected using an enhanced Amersham ECL chemiluminescence kit (GE Healthcare Life Sciences, USA) according to the manufacturer's instructions.
β-actin (mouse monoclonal, 1:3000, Abcam) served as the loading control. The intensity of bands was evaluated using a
C-DiGit™ Blot Scanner (LI-COR Biotechnology, USA). Protein levels were measured in two independent experiments.
Lněničková K., Skálová L., Stuchlíková L., Szotáková B, & Matoušková P. (2018). Induction of xenobiotic-metabolizing enzymes in hepatocytes by beta-naphthoflavone: Time-dependent changes in activities, protein and mRNA levels. Acta pharmaceutica (Zagreb, Croatia), 68(1).
