M per extraction reagent

Total cell lysates of HepG2 and cultured mouse hepatocytes cells and liver homogenates were extracted using M-PER Extraction Reagents (Thermo Fisher Scientific, Waltham, MA), as described.6 (link) The antibodies used in this study are listed in Supplemental Table 2 (http://links.lww.com/HC9/A133).

The cells (5×10⁴ cells/well) were incubated with AH (0, 20, 40, and 80 μg/ml) for 1 h, followed by incubation in the presence of TNF-α (30 ng/ml). After 1 h incubation, the cells were washed with PBS and protein was collected by using M-PER Extraction Reagents (Thermo Scientific) containing protease and phosphatase inhibitor cocktail. The expression levels of p-IκB-α, p-p65NF-κB and β-actin (loading control) were determined using immunoblotting as described above.

All standard tissue culture reagents, including MCDB 131 medium, fetal bovine serum (FBS), and Penicillin-Streptomycin-Glutamine (100×), were obtained from Life Technologies (Paisley, UK). cOmplete™ EDTA-free Protease Inhibitor Cocktail was purchased from Roche (Basilea, Switzerland). TGF-β1 and TGF-β2 were obtained from R&D (Minneapolis, MN, USA). Enhanced Chemiluminescence (ECL) Western blotting substrate, M-PER Extraction Reagents, and NE-PER Nuclear and Cytoplasmic Extraction Kit were purchased from Thermo Scientific Pierce (Rockford, IL, USA). Small interfering RNA (siRNA) oligonucleotides were purchased from Dharmacon (Lafayette, CO, USA). Epithelial-Mesenchymal Transition (EMT were obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-mouse antibodies, anti-rabbit antibodies conjugated with horseradish peroxidase, and mouse anti-GAPDH, -MRTF-A, and -MRTF-B were from Santa Cruz Biotech (Santa Cruz, CA, USA). X-fect was purchased from Clontech (Mountain View, CA, USA). Bradford, 30% acrylamide/bis 37.5:1 solution, ammonium persulfate (APS), 1,2-bis (dimethylamino) ethane (TEMED), glycine, and blotting membranes were obtained from Bio-Rad (Munich, Germany). Matrigel^TM was from Corning (Tewksbury, MA, USA). All other chemicals and solvents (including H₂O₂) were of the highest analytical grade and were purchased from Sigma-Aldrich (Steinheim, Germany).