Rabbit polyclonal anti phospho p53

Whole-cell lysates were obtained using the ProteoJET™ Mammalian Cell Lysis Reagent (Thermo Scientific, Rockford, IL, USA). Proteins (50 μg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membrane was 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 membrane was then incubated with primary antibody in the same concentration of milk in TBST for 1 h at room temperature, washed three times with TBST for 15 min, and then incubated with the HRP-conjugated secondary antibody at room temperature for 1 h. The membrane was again 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 polyclonal anti-Phospho-p53, mouse polyclonal anti-GAPDH, mouse polyclonal anti-ACTIN (Cell Signaling Inc. Danvers, MA, USA); secondary antibodies (goat-anti-rabbit and goat-anti-mouse) conjugated to horseradish peroxidase (Santa Cruz. CA, USA).