Coro1a

Immunohistochemistry was conducted on thin Sects. (5 μm) of formalin-fixed and paraffin-embedded tissues. The sections underwent deparaffinization in xylene and rehydration using a descending series of ethanol. Tissue antigens were repaired by microwave heating, followed by incubation with 10% normal goat serum to block nonspecific reactions at room temperature for 10 min. Monoclonal rabbit primary antibodies against CD3G (1:200; Abcam, UK), CORO1A (1:200; Abcam, UK), FCGR2A (1:200; Abcam, UK), and GZMH (1:200; Abcam, UK) were applied separately and incubated overnight at 4 °C. Biotin-labeled goat anti-mouse/rabbit IgG and streptavidin-peroxidase (UltraSensitiveTM SP IHC Kit; Maxim, China) were subsequently used. The sections were then developed using diaminobenzidine substrate.

The proximity ligation assay (PLA) were carried out using Duolink In Situ PLA reagents according to the manufacturer's instructions (Sigma) by using the primary antibodies (Coro1a, Abcam; Rab7, Abcam; GDI2, Abclonal; RILP, Proteintech). Samples were imaged using an Olympus IX83‐FV3000 confocal microscope (Olympus). Images were analysed with ImageJ software.

For western blotting (WB) assay, total cells and EVs were washed with ice‐cold PBS and lysed in SDS buffer on ice and boiled for 10 min at 100°C. Then, the samples were resolved by SDS‐PAGE followed by transfer onto PVDF membranes (Millipore) and probed with the indicated primary antibodies (CD63, Abclonal; Alix, Proteintech; Tsg101, Abclonal; CD81, Affinity; Coro1a, Abcam; GM130, Abcam; NEDD8, Abcam; Ub, CST; UBA3, Abcam; Cullin3, Sangon Biotech; UBE2F, Bioss; UBE2M, Abcam; TRIM4, Abclonal; Monla, Abclonal; Monlb, Abclonal; Rab7, Abcam; β‐Actin, Abclonal) and second antibodies (Goat anti‐mouse IgG HRP, Goat anti‐rabbit IgG HRP, MultiSciences).
For immunoprecipitation (IP) assays, cells were lysed in Co‐IP lysis buffer [50 mM Tris‐HCl, 5 mM EDTA, 150 mM NaCl, 0.5% (v/v) Nonidet‐P40, and 10% (v/v) glycerol (pH 7.4), supplemented with 1 mM PMSF, 1 mM Na3VO4, and 10 mM NaF]. The lysate was incubated with M2‐Flag beads (Sigma), anti‐His beads (MBL), anti‐HA beads (MBL) or anti‐Myc beads (Thermo Scientific) overnight at 4°C. The immunoprecipitates were washed at least three times in lysis buffer and then analysed by WB with the indicated antibodies (Flag, CST; His, MBL; HA, CST; Myc, Abmart).