Anti srebp 1c

The Western blot was performed as previously described [25 (link)]. Liver protein extracts were obtained by lyses homogenized tissue in Ripa buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with protease inhibitors (Roche Diagnostics, Mannheim, Germany). Protein samples (40 μg) were resolved on 10% or 12% Tris-HCl polyacrylamide gels and subsequently transferred to a nitrocellulose membrane. Membranes were probed with commercially available rabbit polyclonal anti-ApoE (1:500 dilution), anti-GRP-78 (1:500) (Abcam, Cambridge, MA, USA), anti-β-tubulin (1:200) (Cell Signaling, Danvers, MA, USA), anti- ERP29 and anti-α-tubulin (1:2000), anti-SREBP -1c and anti-SOD2 (1:500) antibodies (Abcam, Cambridge, MA, USA), followed by HRP-conjugated anti-rabbit antibody (1:10000) and ECL Plus detection reagents (GE Biosciences, Piscataway, NJ, USA). Relative ApoE, GRP-78, β-tubulin, α-tubulin, ERP29, SREBP and SOD2 band densities were determined by densitometrical analysis using the Image Studio Lite software from LI-COR Corporate Offices-US (Lincoln, Nebraska USA). In all instances, density values of bands were corrected by subtraction of the background values. The results were expressed as the ratio of ApoE, GRP-78, ERP29 and SOD2 to that of β-tubulin and SREBP to that of α-tubulin our β-tubulin.

The protein expression in the liver was determined through Western blotting following a previously described protocol [20 (link)]. The membranes were then incubated with primary antibody (PPARγ, IL-6, IL-1β, SIRT1, LXRα, pAMPKα, AMPKα, PPARα, pACC, ACC, FAS, SREBP1c, CPT1B, and β-actin) at room temperature for 2 h. In this study, the primary antibodies were anti-PPARγ (Cat# 2435S, 1:1000, Cell Signaling, Danvers, MA, USA), anti-IL-6 (Cat# 21865-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-IL-1β (Cat# 16806-1-AP, 1:1000, Proteintech), anti-SIRT1 (Cat# 9475S, 1:1000, Cell Signaling), anti-LXRα (Cat# ab176323, 1:1000, Abcam, Cambridge, UK), anti-pAMPKα (Cat# AF3423, 1:1000, Affinity, San Francisco, CA, USA), anti-AMPKα (Cat# AF6423, 1:1000, Affinity), anti-PPARα (Cat# sc-398394, 1:500, Santa Cruz, Santa Cruz, CA, USA), anti-pACC (Cat# D7D11,1:2000, Cell Signaling), anti-ACC (Cat# C83B10, 1:2000, Cell Signaling), anti-FAS (Cat# C20G5, 1:2000, Cell Signaling), anti-SREBP1c (Cat# ab28481, 1:2000, Abcam), anti-CPT1B(Cat# DF3904, 1:500, Affinity), and anti-β-actin (Cat# GTX109639, 1:10000, Genentech, San Francisco, CA, USA). The relative expression of proteins was quantified densitometrically using ImageJ software (Wayne Rasband, Madison, WI, USA), and β-actin was used as the internal control.

The liver tissues were homogenized in a protein lysis buffer for 20 min on ice and centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was collected and the protein concentration was measured by the Bradford protein assay. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out with 8–12% polyacrylamide gels at 100 V for 1 h. The resolved proteins were transferred from the gel onto a nitrocellulose membrane (Millipore, Billerica, MA, USA) and probed with anti-SREBP-1c, anti-FAS, and anti-CHREBP (Abcam, Cambridge, MA, USA), anti-ACC and anti-SCD-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3 and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA, USA). This was followed by a secondary antibody conjugated to horseradish peroxidase (1:1000) and determined with enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ, USA). The signal intensity was quantified by an image analyzer (Chemidoc XRS+ system; Bio-Rad Laboratories, Hercules, CA, USA).