Rabbit monoclonal anti ago2

Whole-cell lysates were obtained using the ProteoJET Mammalian Cell Lysis Reagent (Thermo Scientific, Rockford, IL, USA). Proteins (50 μg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk in TBST (50 mM Tris (pH 7.5), 200 mM NaCl, 0.05% Tween 20) at room temperature for 1 h. The membranes were then incubated with the primary antibody in the blocking solution for 1 h at room temperature, washed three times with TBST for 15 min, incubated with the HRP-conjugated secondary antibody at room temperature for 1 h and then washed three times with TBST. Antigen-antibody complexes were detected using the SuperSignal detection reagents (Thermo Scientific, Rockford, IL, USA). The following antibodies were used: rabbit monoclonal anti-p53, rabbit monoclonal anti-AGO2, rabbit monoclonal anti-GADD45A, mouse monoclonal anti-GAPDH (Cell Signalling Technology, MA, USA) and horseradish peroxidase-conjugated goat-anti-rabbit and goat-anti-mouse secondary antibodies (Santa Cruz. CA, USA).