Rabbit anti flag ae004

Cell samples or EV samples were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Calbiochem, CA, USA) and phosphatase inhibitors (Calbiochem). The total protein concentration was determined using a BCA Protein assay kit. Equal amounts of proteins were electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes (PVDF, Millipore, USA). The PVDF membrane was blocked with 5% skim milk buffer and then incubated with one of the following primary antibodies: mouse anti-HSP70 (ab2787, Abcam, Cambridge, MA, USA), rabbit anti-TSG101 (14497–1-AP, Proteintech, Rosemont, IL, USA), rabbit anti-Alix (12422–1-AP, Proteintech), rabbit anti-GM130 (12480, Cell Signaling Technology, Beverly, MA, USA), rabbit anti-HGF (26881–1-AP, Proteintech), rabbit anti-IL-10 (20850–1-AP, Proteintech), rabbit anti-SOD2 (24127–1-AP, Proteintech), rabbit anti-TGF-β (21891–1-AP, Proteintech), mouse anti-ACE2 (66699–1-Ig, Proteintech), and rabbit anti-Flag (AE004, ABclonal Biotech Co., Ltd., Cambridge, MA, USA) overnight at 4 °C. The PVDF membranes were incubated with HRP-conjugated secondary antibodies at 37 °C for 1 h. The protein bands on the PVDF membranes were observed using an enhanced chemiluminescence kit (Millipore) and quantified using ImageJ software (NIH, Bethesda, MD, USA).