【出版期刊】《International Journal of Biochemistry and Cell Biology》
【产品原文引用】
2.8. Western blot
The radioimmunoprecipitation assay (RIPA) lysis buffer was used to extract total protein from cells and protein concentrations were quantified using the Bradford method. Samples (30 μg each) of extracted protein were loaded onto sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, separated using electrophoresis, then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were subsequently incubated with anti-USP1 (Fine biotech, Wuhan, China), c-Myc (Huabio, Hangzhou, China), Bad (Bioyotime Technology, Shanghai, China), Bax (Biolab Technology, Xian, China), Bcl-2 (Hengyuan Biological, Shanghai, China), phosphoinositide 3-kinase (PI3K; Biolab Technology, Xian, China), phospho-PI3K (p-PI3K; Bioworld Technology, Bloomington, USA), caspase-3 (Biolab Technology, Xian, China), E-cadherin (Huabio, Hangzhou, China), Akt (Huabio, Hangzhou, China), or p-Akt (Bioworld Technology, Bloomington, USA) primary antibodies overnight at 4 ?C, followed by incubation with a secondary antibody (Abcam) conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. The membranes were then briefly incubated with an enhanced chemiluminescence (ECL) solution to visualize the protein band of interest.
