Mouse monoclonal antibody against gapdh

Proteins were extracted by lysing cells in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris–HCl [pH 6.8], 2% SDS) containing 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL pepstatin, 12.5 μg/mL leupeptin, 2 μg/mL aprotinin, 1 mM sodium orthovanadate, and 1 mM sodium molybdate. Cell extracts were processed for western immunoblotting as described previously [50 (link)]. The following antibodies used for immunoblotting were purchased from the indicated suppliers: mouse monoclonal antibody against AF1q from Abnova (Taipei City, Taiwan), mouse monoclonal antibody against GAPDH from Sigma Aldrich (St Louis, MO), mouse monoclonal antibody against cytokeratin 8/18 from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit polyclonal antibody against Fibronectin from Sigma Aldrich, mouse monoclonal antibody against Vimentin from Abcam (Cambridge, UK), rabbit polyclonal antibody against p38 from Santa Cruz Biotechnology, rabbit polyclonal antibody against Thr180/Tyr182 p38 phosphorylations from Cell Signaling (Danvers, MA), rabbit polyclonal antibody against Erk1/2 from Sigma Aldrich, rabbit polyclonal antibody against Thr202/Tyr204 Erk1/2 phosphorylations from Cell Signaling.

Western blot analyses were performed as per the manufacturer's suggestions (Invitrogen, Carlsbad, CA). Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO). The molecular sizes for MCP-1, TLR2, TLR4 and GAPDH are 8.8 kDa, 84 kDa, 96 kDa and 36 kDa, respectively.

Total protein from transduced cells was extracted using RIPA lysis buffer containing a protease inhibitor. 100 μg total lysate were resolved on 7.5% SDS-PAGE. A mouse monoclonal antibody against HIF-1α (Novus Biologicals, Littleton, CO) at 1:1000 dilution, a rabbit polyclonal antibody against HIF-2α (Novus Biologicals) at 1:500 dilution, a rabbit polyclonal antibody against Lipin 1 (Cell signaling, Danvers, MA) at 1:1000 dilution, and a mouse monoclonal antibody against GAPDH (Sigma) at 1:10000 dilution were used. Appropriate horseradish peroxidase–conjugated secondary antibodies, either anti-mouse or anti-rabbit (GE Healthcare, Piscataway, NJ), were used at 1:2500 dilution. Immunoblots were developed using the SuperSignal West Pico chemiluminescent substrate kit (Thermo Scientific, Rockford, IL).