4.5. Western Blotting

Total lysates of Raw 264.7 cells collected from different experiments were prepared using RIPA lysis and extraction buffer (#89901, ThermoFisher Scientific, Waltham, MA, USA) to which cOmplete™ Protease Inhibitor Cocktail (#11697498001, Millipore Sigma, St. Louis, MO, USA) and PhosSTOP phosphatase inhibitor (#4906845001, Millipore Sigma, St. Louis, MO, USA) were freshly added. Extraction of separate cytoplasmic and nuclear protein fractions were prepared using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (#78833, ThermoFisher Scientific, Waltham, MA, USA) to which cOmplete™ Protease Inhibitor Cocktail was added. Protein concentrations were measured using Pierce BCA Protein Assay Kit (#23225, ThermoFisher Scientific, Waltham, MA, USA). Equal amounts of protein lysates (20 μg) were separated using gradient 4–12% NuPAGE Bis-Tris gels and transferred to nitrocellulose membranes using Power Blotter pre-cut membranes and filters (#PB7320, ThermoFisher Scientific, Waltham, MA, USA). Following transfer, membranes were blocked for 1 h using 5% Blotting Grade Blocker Non-Fat Dry Milk (#1706404, Bio-Rad, Hercules, CA, USA). Membranes were incubated with appropriate primary antibody overnight at 4 °C. Primary antibodies NFATc1 (#sc-7294), c-fos (#sc-166940) (Santa Cruz Biotechnology, Dallas, TX, USA), AhR (#AF6697, R&D Systems, Minneapolis, MN, USA), Histone H3 (#4499T) and GAPDH (#2218S) (Cell signaling, Danvers, MA, USA) and ß-actin (#A5441, Millipore sigma, St. Louis, MO, USA) were used. Membranes were washed 3 times for 10–15 min and then incubated with appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Membranes were washed 3 times for 10–15 min and visualized using Pierce ECL detection system (#32106, ThermoFisher Scientific, Waltham, MA, USA) on Amersham Imager 600 (GE Healthcare, Pittsburgh, PA, USA). The intensity of immunoreactive bands was quantified using Image Lab (Bio-Rad, Hercules, CA, USA).