Rabbit polyclonal anti p53

The total protein of INS-1 cells and mouse pancreatic islets were extracted by using a RIPA buffer. Nuclei proteins were extracted from the cells by using the Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce, USA). The protein concentrations were then determined by a micro BCA assay. The total protein was separated on a 4–12% (wt/vol) SDS-PAGE. After that, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA). The membrane was blocked with 5% skimmed milk before being incubated overnight at 4 °C with one of the following primary antibodies: rabbit polyclonal anti-BTG₂ (Santa Cruz Biotechnology, USA), rabbit polyclonal anti-p53 (Santa Cruz Biotechnology, USA), rabbit polyclonal anti-Bax (Santa Cruz Biotechnology, USA), or mouse monoclonal anti-β-Actin (Santa Cruz Biotechnology, USA). After washing, the membrane was incubated with one of the following secondary antibodies: horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, USA), or horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, USA), at room temperature. The protein bands were detected with an enhanced chemiluminescence system (Pierce Biotechnologies, USA) and exposed on x-ray films. The band intensities of proteins were quantified by using ImageJ v 1.43 software. All Western blot results were shown in supplement data.

The Western blotting procedure was performed as described before [55 (link)]. The following antibodies and dilutions were used: rabbit polyclonal anti-p53 (1:200, Santa Cruz Biotechnology, cat. sc-6243, Dallas, TX, USA), rabbit monoclonal anti-p21 (1:1000, Cell Signaling Technology, CST, cat. 2947, Danvers, MA, USA), rabbit monoclonal anti-MDM2 (1:1000, Thermo Fisher Scientific, cat. 700555, Waltham, MA, USA), rabbit monoclonal anti-GAPDH (1:4000, CST, cat. 2118, Danvers, MA, USA) and goat peroxidase-conjugated anti-rabbit (1:2000, CST, cat. 7074, Danvers, MA, USA).