Anti srebp 1 sc 366

Nuclear and cytoplasmic extracts were prepared from liver tissue and cultured cells using a commercially available nuclear extraction kit (Cayman Chemical Co., Ann Arbor, MI, USA). Twenty micrograms of protein was resolved on a 4%-12% Bis-Tris NuPAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot Transfer Stack (Invitrogen). After protein transfer, the following primary antibodies were used: anti-phospho-GSK-3β (#9323; Cell Signaling Technology, Danvers, MA, USA), anti-GSK-3β (#9315; Cell Signaling Technology, Danvers, MA, USA), anti-β-catenin (#9582; Cell Signaling Technology, Danvers, MA, USA), anti-TCF4 (#2569; Cell Signaling Technology), anti-PPARγ (#2443; Cell Signaling Technology), anti-SREBP-1 (sc-366; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-β-actin (#4967; Cell Signaling Technology), and anti-Lamin B1 (ab16048; Abcam, Cambridge, MA, USA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected by chemiluminescence (GE Healthcare, Piscataway, NJ, USA).

The cells or liver tissues were lysed with RIPA buffer (Solarbio, Beijing, China). Fifteen μg of protein was separated by 10% SDS-PAGE and further transferred onto a PVDF membrane (Millipore, Boston, Massachusetts, USA). The membrane was blocked with 8% non-fat dry milk and incubated with primary antibodies at 4°C overnight. The specific primary antibodies used in this study were anti-SREBP1 (sc-366) (Santa Cruz, Dallas, Texas, USA), anti-FAS (#3180) (Cell Signaling, Boston, Massachusetts, USA), anti-β-actin (#3700) (Cell Signaling, Boston, Massachusetts, USA) and anti-JUN (#9165) (Cell Signaling, Boston, Massachusetts, USA). After washing three times with PBST, the membrane was incubated with the HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Zhongshanjinqiao, Beijing, China. 1:5000) for 2 h at room temperature. Immunodetection was conducted using the ECL Plus detection system (Millpore, Boston, Massachusetts, USA) according to the manufacturer's instructions.