Horseradish peroxidase conjugated anti rabbit immunoglobulin g igg anti mouse igg

Cells and tissues were harvested in sampling buffer [62.5 mmol/L Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS)] and heated for 5 minutes at 100°C. Protein concentration was determined with the Bradford assay using a commercial kit purchased from Bio-Rad Laboratories (Hercules, CA, USA). Equal quantities of protein were separated electrophoretically on 10% SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The membranes were probed with diluted antibody. The expression of target proteins was determined with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG)/anti-mouse IgG (Sigma-Aldrich, St Louis, MO, USA) and enhanced chemiluminescence (Pierce, Rockford, IL, USA) according to the manufacturers' suggested protocols. The membranes were stripped and reprobed with an anti-β-actin mouse monoclonal antibody (Sigma-Aldrich) as a loading control. The related antibodies were anti-phosphorylated(p)-Smad3, anti-E-cadhernin, anti-Vimentin, anti-β-catenin (Cell Signaling Technology, Beverly, MA, USA), anti-CD24, anti-CD44, anti-CD166, anti-EpCam, anti-EF-1, anti-p84 anti-FRAT2, anti-LRP6, anti-FZD4, anti-FZD5 (Abcam, Cambridge, MA, USA) and anti-CD133 (Epitomics, USA).

Cells and tissues were harvested in sampling buffer [62.5 mmol/L Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS)] and heated for 5 minutes at 100°C. Protein concentration was determined with the Bradford assay using a commercial kit purchased from Bio-Rad Laboratories (Hercules, CA, USA). Equal quantities of protein were separated electrophoretically on 10% SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The membranes were probed with diluted antibody. The expression of target proteins was determined with horseradish peroxidase–conjugated anti-rabbit immunoglobulin G (IgG)/anti-mouse IgG (Sigma-Aldrich, St Louis, MO, USA) and enhanced chemiluminescence (Pierce, Rockford, IL, USA) according to the manufacturers’ suggested protocols. The membranes were stripped and reprobed with an anti–β-actin mouse monoclonal antibody (Sigma-Aldrich) as a loading control. The related antibodies were anti-Smad2/3, anti–NF-κB inhibitor (IκBα), anti–matrix metalloproteinase 9 (MMP9), anti–phosphorylated (p)-Smad2, anti–p-Smad3, anti-Smad2, anti–vascular endothelial growth factor (VEGF) (Cell Signaling Technology, Beverly, MA, USA), anti-QKI, and anti–green fluorescent protein (GFP) (Abcam, Cambridge, MA, USA).