Enhanced chemiluminescence western blot detection system

The liver tissues were homogenized in 500 μl of a RIPA buffer (Sigma-Aldrich, St. Loius, MO, USA) for protein extraction. The protein (40 μg) was then separated on a 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene difluoride membranes. The membranes were incubated in a blocking solution (5% bovine serum albumin) at room temperature for 2 h and then incubated with a primary antibody (OB-R, SREBP-1, FAS, JAK2, AMPK, ACC, STAT3 or β-actin) overnight at 4 °C. After washing, the membranes were incubated with a goat anti-rabbit immunoglobulin G peroxidase-conjugated secondary antibody. The membranes were developed using an enhanced chemiluminescence western blot detection system (GE Healthcare Biosciences, Pittsburgh, PA, USA) as described previously.^{12 , 23 (link), 24 (link)}

Total protein of cells was extracted by radioimmunoprecipitation assay (RIPA) lysis buffer (Cat: R0010) with phenylmethanesulfonyl fluoride (Cat: ST506), and quantified by BCA protein assay kit (Cat: 10136–1). The β‐tubulin antibody (Lot: 334077) was used as the loading control for Western blot analysis to confirm equivalent protein loading in different groups. Same amounts of proteins derived from cell lysates were resolved by SDS‐PAGE and transferred onto nitrocellulose membranes. Nonspecific binding was blocked by 5% skim milk in phosphate buffer saline at room temperature for 2 h. After washing three times with tris‐buffered saline Tween‐20 (TBST), the membranes were incubated with rabbit anti‐NMDA‐2A antibody (Lot: 00083510) diluted 1:800 in TBST and rabbit anti‐tubulin antibody (Lot: 334077) diluted 1:1000 in TBST at 4°C overnight. In addition, the membranes were washed three times with TBST and incubated with corresponding secondary antibodies conjugated with horseradish peroxidase at room temperature for another 2 h. Enhanced chemiluminescence Western blot detection system (GE Healthcare) was utilized to visualize antibody‐binding conjugates. Image‐J software (1.8.0) was used to quantify the bands.

Western blot analysis was performed on proteins (10–30 μg) isolated from whole cell lysates, as described previously. The following primary antibodies were used: rabbit polyclonal anti-APJ (1:1000; ab84296, Abcam), rabbit monoclonal anti-PDGFR-β (1:10,000; ab32570, Abcam), and rabbit polyclonal anti-β-actin (1:5,000; NB600-532H, Novus Biologicals). Corresponding horseradish peroxidase-conjugated secondary antibodies (DAKO) were subsequently applied. Signals were visualized with an enhanced chemiluminescence western blot detection system (GE Healthcare UK Ltd).