Ab85047

Total protein of PDAC cells was extracted by RIPA lysis buffer (Thermo, USA) containing protease inhibitor cocktail tablets (Roche, USA) and exosomal protein was isolated using Exosome RNA and Protein Isolation Kit (Thermo, USA). After measured by BCA Protein Assay Kit (Beyotime, China), equal amounts of protein (30 μg) were used to SDS-page on 10% polyacrylamide gels and transferred to PVDF membranes (Millipore, USA), which were blocked and blotted with primary antibodies overnight at 4℃. The antibodies used in this study included the following: anti-p27 (1:800, #3686, CST, USA), anti-p-p27 (1:1000, ab85047, abcam, USA), anti-PPP2R2A (1:1000, #5689, CST, USA), anti-Histone H1(1:1000, 15446-1-AP, Proteintech, USA), anti-β-actin (1:5000, 20536-1-AP, Proteintech, USA), anti-AKT (1:1000, 4691, Cell Signaling, USA), antip-AKT (1:1000, 4060, Cell Signaling, USA) and anti-CD63 (1:500, 10628D, Thermo, USA). The membranes were washed with PBST and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h and the immunocomplexes were then visualized using a New Super ECL Detection Kit (KeyGEN BioTECH, China) according to the manufacturer's protocol.