Ecl hyper film

Cellular or EV proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with the respective primary antibody against CD63 (1:1000; Abcam), beta-actin (1:20000; CST), ALIX (1:1000; CST), flotillin-1 (1:1000, CST), RAB27A (1:1000; Abcam), ETA (1:2000; Abcam), Akt (1:2000; CST), phospho-Akt (1:2000; CST), or PD-L1 (1:1000; CST). After removing the primary antibody and three washing steps at 10-min intervals, the blots were incubated with a horseradish peroxidase (HRP)-linked secondary antibody. The images were visualized using enhanced chemiluminescence (ECL) detection reagents (Thermo Scientific) and quantified using ECL hyper-film (AGFA) and Fusion FX7 system (Vilber Lourmat).

The concentration of cellular or sEV proteins was determined using a bicinchoninic acid (BCA) assay kit (#23227, Thermo Scientific, Waltham, MA, USA) or micro BCA assay kit (#23235, Thermo Scientific). The proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes, bound with primary antibodies, and incubated with horseradish peroxidase (HRP)-linked secondary antibody. Images were visualized using enhanced chemiluminescence (ECL) detection reagents (#34580, Thermo Scientific) with ECL hyper film (AGFA, Morstel, Belgium) and a Fusion FX7 system (Vilber Lourmat, Eberhardzell, Germany). Western blots were normalized to β-actin in the whole cell lysate and CD63 in the proteins of sEV. Densitometric analysis was performed using image J. The following primary antibodies were used: PD-L1 (#13684, CST), Flotilin-1 (#3253, CST), TSG101 (ab30871, Abcam, Cambridge, UK), CD63 (ab68418, Abcam), Alix (#2171, CST), Syntenin-1 (ab133267, Abcam), Rab11 (ab18211, Abcam), Rab7 (ab50533, Abcam), Rab27A (ab55667, Abcam), Rab27B (ab76779, Abcam), LC3 (NB600-1384, Novus, Stroudsburg, PA, USA), p62 (#5114, CST), Beclin-1 (#3738, CST), and β-actin (SC-47778, Santa Cruz Biotechnology, Dallas, TX, USA). All the whole western blot figures can be found in the Supplementary Materials (Figure S1).