Fluorchem hd2 gel documentation system

To determine the expression of CYP1A1 and CYP1B1 enzymes, equal amount of protein (20 μg) obtained from vehicle- and CSC-treated macrophages was loaded on a 10% polyacrylamide gel and electrophoresed, followed by transfer to a PVDF membrane. The membrane was blocked in 5% non-fat dry milk and incubated with primary antibody (1:500–1000 dilution) overnight. Next day, the membrane was washed and incubated with secondary antibody (1:2000 dilution). After washing, the blot was visualized by Luminata crescendo western HRP substrate using the Alpha Innotech FluorChem HD2 gel documentation system (Alpha Innotech, San Leandro, CA), and the densitometric data was analyzed using AlphaEase FC StandAlone software (version 6.0.0.14; Alpha Innotech). Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was used as an internal loading control to normalize the expression of CYP1A1 and CYP1B1 proteins.

Cell pellets were harvested and lysed using Triton-X-100 lysis buffer (1% triton X-100, 150 mM NaCl, 25 mM Tris pH 7.5). Approximately 50 μg of total cellular protein from each sample were subjected to SDS-PAGE, proteins were transferred to nitrocellulose membranes, and the membranes were blocked with 5% nonfat milk in a Tris-buffered saline solution containing 0.1% Tween-20 for 1 h. The blots were then probed overnight with relevant antibodies, washed, and probed with species-specific secondary antibodies coupled to horseradish peroxidase. Immunoreactive material was detected by enhanced chemiluminescence (Protein Simple, Santa Clara, CA). Densitometry analysis to quantify band intensity was performed using an Alpha Innotech FluorChem HD2 gel documentation system (Alpha Innotech, Santa Clara, CA).