Ripa lysate

RIPA lysate (Cat # KGP702, KeyGen, Nanjing, China) was used to extract the total protein from cells and frozen tissues which was quantified through BCA (Bicinchoninic acid) protein quantitative kit (Cat # KGP902, KeyGen) and electrophoresed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). PVDF (Polyvinylidene fluoride) membranes (Cat # 162–0177, Bio-Rad, Hercules, CA, USA) were used for protein transfer and then incubated with 5% nonfat milk at room temperature for 1.5 h. The membranes were incubated with primary antibody at 4°C overnight. After being washed with TBST (Tris-Buffered Saline and Tween 20) for three times, the membranes were added with secondary antibody and incubated at room temperature for 1 h. After then, TBST was used to wash the membranes for three times. Protein expression was detected using ECL (Electrochemiluminescence) detecting reagents (Cat # KGP1121, KeyGen).

Total protein was extracted with RIPA lysate (KeyGEN BioTECH, Nanjing) and PMSF (protease inhibitor, KeyGEN BioTECH, Nanjing). The lysates were centrifuged at 12 000 rpm for 20 min and the supernatants were collected. Samples (10 μl) were run on a SDS-PAGE gel followed by blotting to a nitrocellulose membrane. Membranes were blocked with 5% skim milk for 2 h at room temperature, then the membranes were incubated overnight at 4°C with the following primary antibodies: β-actin (1: 4000 Proteintech, USA), PTEN (1: 10000 Abcam, USA), and PI3K (1: 2000 Cell Signaling TECHNOLOGY, USA). Before adding secondary antibody (1: 10 000 PROMEGA, USA), we washed the membranes with TBST 3 times followed by incubation for 2 h at room temperature. Rhea ECL (US EVERBRIGHT INC, Suzhou) was used as developer reagent and the band intensity was assessed using Image J lab software.