Mouse anti β actin

Western blotting was performed as previously described [54 (link)]. After incubation with the primary antibodies, including anti-rabbit Myocardin (Sigma, United States), anti-rabbit MRTF-A (Abcam, United States), anti-rabbit SRF (Santa Cruz, United States), anti-rabbit ACTA2 (Abcam, United States), anti-rabbit SM-22 (Abcam, United States), anti-rabbit OPN (Santa Cruz, United States), anti-rabbit CyclinD1 (Santa Cruz, United States), anti-Rabbit CYR61 (Santa Cruz, United States), anti-rabbit MYL9 (Santa Cruz, United States), anti-rabbit LC3 (Novus Biologicals, United States), anti-mouse P62 (BD Transduction Laboratories, United States), anti-rabbit ATG7 (Bioss, China) and anti-mouse β-actin (Santa Cruz, United States) antibodies; the membranes were incubated with the appropriate secondary antibodies, including IR Dye-800 conjugated anti-rabbit IgG secondary antibody and IR Dye-680 conjugated anti-mouse IgG. The specific protein was visualized using the Odyssey Infrared Imaging System (Gene Company, HongKong). β-actin expression was used as an internal control to show equal loading of the protein sample. The relative quantity of proteins was analyzed using Image J software. The relative quantity of proteins was analyzed using Image J software.

Liver tissues were stored at −70 °C. Tissue samples were homogenized in ice-cold RIPA lysis buffer (Millipore, Billerica, MA, USA) for protein extraction. Tissue debris was removed by centrifugation, and the resulting supernatants were collected and analyzed for protein concentration by the BCA protein assay kit (Thermo Scientific, Bartlesville, OK, USA). The protein was separated on a 10% SDS polyacrylamide gel and then transferred to nitrocellulose membranes (Hybond, GE Healthcare Life Sciences, Little Chalfont, UK). The membranes were incubated with specific primary antibodies overnight at 4 °C. The primary antibodies included anti-mouse β-actin, anti-mouse PPAR-α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-rabbit TNF-α, anti-rabbit PPAR-γ, and anti-rabbit GLUT4 (Cell Signaling Technology, Inc., Beverly, MA, USA). After washing, the membranes were allowed to react with diluted horseradish peroxidase-conjugated secondary antibodies, including goat anti-rabbit IgG antibody and horse anti-mouse IgG (Cell Signaling Technology, Inc., Beverly, MA, USA) at room temperature for 2 h. An enhanced chemiluminescence system (SuperSignal, Thermo Scientific, Rockford, IL, USA) was used to visualize antibody–antigen complexes.

Total protein was extracted from MC3T3-E1 cells on the Ti discs using a NP-40 lysis buffer. After electrophoresis and transferring protein, the membrane was blotted with the primary antibodies for 16 h at 4°C, such as 1:2000 of anti-rabbit Runx-2 (SantaCruz Biotechnology Inc., Dallas, TX, USA), 1:2000 of anti-goat OPG (SantaCruz Biotechnology Inc.), 1:1000 of anti-mouse RANKL (Novus Biological Inc., Centennial, CO, USA) and 1:2,500 of anti-mouse β-actin (Santa Cruz Biotechnology Inc.). After washing, the membrane was blotted with 1:5000 of either horseradish peroxidase (HRP)-conjugated goat anti-rabbit or mouse-IgG (Enzo Life Sciences Inc., New York, NY, USA) and HRP- conjugated donkey anti-goat-IgG (SantaCruz Biotechnology Inc.). The developing was performed using X-ray film (Fuji Film Co.) after detection using an ECL solution (Merck Millipore, Burlington, MA, USA). The density of the expressed bands was measured using Science Lab Image Gauge (Fuji Film Co.).

Cells were washed with cold PBS and lysed in lysis buffer containing: 50mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM Na₃VO₄, 1 mM NaF, 0.25% sodium deoxycholate, 1% NP40, 1 mM EGTA, 1 mM PMSF and protein inhibitor cocktail from Sigma–Aldrich (P8340). Protein concentration was calculated with Bradford assay (Bio-Rad, Hercules, CA, United States). Eighty microgram of cell lysate (or 20 μg for human mucosa) was denatured at 90°C for 10 min in 1:1 volume of 2X Laemmli sample buffer (Bio-Rad, United States) and 8% 2-mercaptoethanol. Protein samples were separated on a 10% SDS–PAGE gel and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, United States). After blocking in 5% non-fat dry milk buffer, the membranes were incubated overnight at 4°C with rabbit anti-P2Y₂ (1:200; Alomone, Jerusalem, Israel, APR-010), anti-P2Y₄ (1:800; Alomone, APR-006), anti-P2Y₆ (1:600; Alomone, APR-011), TPH1 (1:1000; Novus Biologicals, Littleton, CO, United States, NB110-57629) or anti-mouse β-actin (1:1000; Santa Cruz Biotech, Dallas, TX, United States, sc-47778) in blocking solution. Membranes were washed and incubated with the HPR-conjugated secondary anti-rabbit or anti-mouse antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States).