Anti srebp 1

Total protein was extracted by RIPA buffer (Solarbio, Beijing, China) containing protease inhibitors (PMSF) (Solarbio, Beijing, China), while nuclear protein was extracted using a Nuclear Protein Extraction kit (Solarbio, Beijing, China). The protein concentrations were determined using a BCA Protein Assay kit (Solarbio, Beijing, China). The tissue lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Pall Co, USA) at a voltage of 90V. Blotted membranes were blocked with 5% BSA for 1.5 h at room temperature and incubated overnight at 4°C with anti-FAS(#3180, Cell Signaling Technology), anti-SREBP-1(#NB100-2215, Novus), anti-NRF2(#12721, Cell Signaling Technology), anti-AHR (#sc-13308, Santa Cruz Biotechnology), anti-Lamin-B2(#12255, Cell Signaling Technology) and anti-β-Actin(#3700, Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:5,000, Zhongshan Golden Bridge Biotechnology, Beijing, China) for 1 h at room temperature. Fluorescent bands were visualized and photographed using an SYNGENE CGQ/D2 GEL-Image System (Bio-Rad, America).

Immunoblotting was performed as previously described39 . The following antibodies were used in this study: anti-CREBH, anti-Insig-1 and anti-Insig-2 (Santa Cruz, USA); anti-SREBP-1 and anti-SREBP-2 (Novus, USA); anti-Flag (Cell signaling, USA). All antibodies were used at a final concentration of 0.1–1 μg/ml. After incubation with the appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (1:5000 dilution; GE Healthcare UK), proteins were visualized by enhanced chemiluminescence (ECL) according to the manufacturer’s instructions (Amersham Biosciences, Pittsburgh, PA, USA).

Samples containing equal amounts of total protein were mixed with gel loading buffer (50 mM Tris, 10% SDS, 10% glycerol, 10% 2-mercaptoethanol, 2 mg/mL bromophenol blue) in a ratio of 4:1 (v/v) and incubated at 95 °C for 5 min. Samples (30 μg of protein) were separated on SDS-polyacrylamide gels (12%) (Mini Protean II, Bio Rad, Hercules, CA, USA) using the Laemmli buffer system and proteins were semidry transferred to nitrocelulose membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked for 1 h with 5% (w/v) nonfat dried milk in TTBS and incubated overnight at 4 °C with specific primary antibodies: anti-SCD1 (1:500, R&D Systems, Minneapolis, MN, USA), anti-SREBP1 (1:1000, Novusbio, Littleton, CO, USA), anti-β-ACTIN (1:5000, Sigma, Saint-Louis, MO, USA) then for 1 h with HRP-conjugated secondary antibodies (GE Healthcare, Chicago, IL, USA). Bands were developed with the use of ECL-system reagents (GE Healthcare, Chicago, IL, USA). Rainbow markers (Amersham Biosciences, Amersham, UK) were used for molecular weight determinations. Protein pattern images were taken using an ImageQuant Las 500 (GE Healthcare, Chicago, IL, USA). Data analysis was performed using Image Lite Studio software (LI-COR, Lincoln, NE, USA).