Immun star western chemiluminescent kit

Total liver microsomal protein concentrations in samples were determined with the Bradford method [20 (link)] using bovine serum albumin (BSA) as standard. Total cellular proteins (50 μg) were separated by 12% precast sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE: Bio-Rad). The gel was blotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and incubated with the primary polyclonal antibodies: CYP1A (Biosense Laboratories 1:500), CYP3A (1:2000), and CYP2E1, CYP2B1/2B2, CYP26 (Santa Cruz Biotechnologies, 1:500) antisera. After washing, membranes were incubated with peroxidase conjugated goat anti-rabbit/mouse antibodies (GAR/GAM-HRP; Bio-Rad) diluted 1:3000, developed using an Immun-Star Western Chemiluminescent Kit (Bio-Rad) and visualized with Eastman Kodak Company’s Molecular Imaging Systems (Rochester, NY, USA).

Total protein extracts from normal, PTC, and PTC with HT samples were prepared using the T-PER tissue protein extraction reagent (Pierce, Rockford, IL, USA) containing a cocktail of protease inhibitors (Pierce, Rockford, IL, USA). Protein concentration was determined with the BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. Proteins were separated by SDS-PAGE in 6–12% polyacrylamide gels and transferred onto nitrocellulose membranes (Millipore Corp, Bedford, MA, USA). Membranes were blocked with Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% non-fat dry milk for 60 min at room temperature. After washing four times with TBST, membranes were incubated with primary antibodies for E-cadherin, pSTAT3 (Cell Signaling Technology, Danvers, MA, USA), TGF-β1, and VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight, washed four times with TBST, and incubated with horseradish peroxidase-conjugated species-specific secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature and then washed again four times. Signals were visualised using the Bio-Rad Molecular Imager ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) with the Immun-Star™ Western™ Chemiluminescent kit (Bio-Rad, Hercules, CA, USA).