Ab227849

Protein was isolated using RIPA lysis buffer (JRDUN, Shanghai, P.R. China), concentration-measured by an enhanced BCA protein assay kit (Thermo Fisher Scientific), separated by 10% SDS-PAGE, and immunoblotted to PVDF membranes (Millipore, Billerica, MA, USA), blocked with 5% nonfat dry milk for 1 h at room temperature, and probed with primary antibodies at 4°C overnight. After washing with PBST, the bolts were incubated with a second antibody for 1 h at 37°C. An enhanced chemiluminescence system (Tanon, Shanghai, P.R. China) was used to visualize protein. Primary antibodies’ information was provided as follows: CD9 (Ab92726, Abcam, St. Louis, MO, USA), CD81 (Ab109201, Abcam), ATG14 (Ab227849, Abcam), and GAPDH (60004-1-1G, Proteintech, UK).

The expressions of Beclin1, RUNX1, ATG14, and GAPDH proteins were detected by western blot. The primary antibodies used included rabbit monoclonal anti-Beclin1 (Abcam, ab210498); rabbit polyclonal RUNX1 (Proteintech, 25,315–1-AP); rabbit polyclonal ATG14 (Abcam, ab227849), and rabbit monoclonal anti-GAPDH (Abcam, ab181602). The band density was analyzed using a gel imaging system and compared with the internal control.