Cd68 primary antibody

Total proteins of cells and exosomes were extracted, and protein concentration was determined using a BCA kit (AmyJet Scientific, Wuhan, Hubei, China). The extracted proteins were mixed with the loading buffer, boiled at 95°C for 10 min, centrifuged, electrophoresed with 10% polyacrylamide gel, and transferred to the membrane, followed by 1-h blocking with 5% skim milk in Tris-buffered saline with Tween 20 (TBST). Then, ATF2 primary antibody (1:1,000, Cell Signaling Technology, Beverly, MA, USA) was added, along with CD68 primary antibody (1:200, Abcam, Cambridge, MA, USA), CD206 primary antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD63 primary antibody (1:200, Santa Cruz Biotechnology), CD81 primary antibody (1:200, Santa Cruz Biotechnology), TSG101 primary antibody (1:1,000, Abcam), GRP49 primary antibody (1:1,000, Abcam), and GAPDH primary antibody (1:1,000, Cell Signaling Technology) for overnight incubation at 4°C. Then, the membranes were developed using enhanced chemiluminescent reagent (Thermo Fisher Scientific). Quantification of signals on western blots was conducted using National Institutes of Health ImageJ imaging and processing analysis software with signaling intensity normalized to GAPDH. The experiment was repeated three times with the data averaged.