Mouse monoclonal antibody specific for gapdh

Total proteins were extracted from cells using radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail. A bicinchoninic acid assay was used to determine the concentrations of proteins in the samples. Accordingly, equal amounts of proteins were loaded onto to sodium dodecyl sulfate–polyacrylamide gels for electrophoretic separation, followed by transfer to nitrocellulose membranes. The membranes were blocked in phosphate-buffered saline with Tween-20 buffer containing 5% fat-free milk and incubated in a 1:200 dilution of a mouse monoclonal primary antibody specific for IGF2BP3 (Santa Cruz Biotechnology, Inc) or a 1:5000 dilution of a mouse monoclonal antibody specific for GAPDH (Santa Cruz Biotechnology, Inc) overnight at 4°C. The membranes were then washed and incubated in a 1:2000 dilution of a horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology, Inc) for 2 hours at room temperature. Finally, the membranes were incubated with an enhanced chemiluminescence reagent (Beijing Applygen Technologies Inc, Beijing, China ) and visualized.