Ecl kit

To evaluate the protein expression levels of M2 expression plasmids and budding efficiency, plasmids were transfected into cultured 293T cells and the supernatant was collected after transfection. Twenty-four hours post transfection, the cells were collected and lysed with RIPA cell lysis buffer (Yeasen). The cell lysates were sent to Western blotting assay by loading onto 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and were then transferred to a PVDF membrane, followed by blocking in PBS buffer containing 5% skimmed milk. The proteins were detected by incubating with mouse anti-FLAG antibody or anti-HA antibody (Sigma-Aldrich, St. Louis, MO, USA) and then with horseradish peroxidase-conjugated anti-mouse antibody (Yeasen); they were then visualized with the ECL kit (Yeasen).

Total protein was extracted by Cell lysis buffer for Western and IP (Beyotime, Shanghai, China) and quantified by the BCA methods. Protein bands were separated by SDS‐PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. Finally, the bands were visualized with an enhanced chemiluminescence (ECL) Kit (Yeasen, Shanghai, China) using an Image Quant LAS 4000 mini (GE). Information on antibodies is shown in Table S1.

Western blotting: Primary antibodies: anti‐CD63 (ab217345, Abcam, USA), anti‐Alix (ab275377, Abcam), anti‐TSG101 (ab125011, Abcam), anti‐Calnexin (ab313243, Abcam). The membranes were developed using ECL kit (Yeasen, China).
TEM: DsEVs sample was examined by TEM (Hitachi TEM system, Japan).
NTA: 10 µL DsEVs suspension was loaded into the sample chamber of ZetaVIEW S/N 252 (Particle Metrix GmbH, Germany). Data analysis was performed with software (ZetaView 8.04.02, Germany).
Detection of the loading rate of TAT_47‐57‐MBP_87‐99A⁹¹ into DsEVs: After co‐culturing the FITC‐labeled peptide with DCs for 24 h, DCs were collected and stained with phalloidin (Actin‐Tracker Red‐594, C2205S, Beyotime) and 4′,6‐diamidino‐2‐phenylindole (DAPI, C1005, Beyotime, China). After the isolation of DsEVs.
DsEVs internalized by DCs and CD4⁺ T cells in vitro: DsEVs were labelled with the Dil (Solarbio life sciences, China) and then co‐cultured with DCs or CD4⁺ T cells for 24 h. Cells were fixed and followed by staining with phalloidin (Actin‐Tracker Green‐488, C2201S, Beyotime) and DAPI (C1005, Beyotime). The images were obtained by scanning confocal fluorescence microscopy.

GSCs from diverse groups were lysed by RIPA buffer containing 1% protease and phosphatase inhibitors (Beyotime Biotechnology, Beijing, China). The proteins were quantified and denatured, separated by SDS-PAGE gel electrophoresis and transferred onto PVDF membranes. The membranes were then incubated with the primary antibody against the target protein at 4 °C overnight. Next, the primary antibody was washed away and the secondary antibody was added and incubated at room temperature for 1 h. Protein bands were visualized using the chemiluminescence ECL kit (YEASEN, Shanghai, China) and a chemiluminescence imaging system (Tanon, Shanghai, China). The gray values of the protein bands were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA). The antibodies used in this study were listed in the Table S4.

Collect the cells from the cell flask into a 1.5 mL EP tube and added RIPA lysis buffer (Beyotime, CHN). The protein lysate (30 μg) was subjected to 10% SDS-PAGE and then electrotransferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore IPVH00010, Solarbio, CHN). The main antibodies are as follows: β-actin (ab6276, Abcam, UK), EDN3 (H00001908-M01, Novus Biologicals, USA), the ratios of them to the primary antibody dilution buffer (Beyotime, CHN) are 1:10000 and 1:1000. The bands were washed the next day and the secondary antibodies were incubated for one hour at room temperature. The ratio of β-actin and EDN3’s secondary antibody (Proteintech, CHN) to the secondary antibody dilution buffer (Beyotime, CHN) is 1:10000 and 1:5000. The bands were washed and soaked in ECL kit (yeasen, CHN) and analyzed by ChemiDoc XRS + image analyzer (Bio-Rad, USA).

The cells were collected and lysed in RIPA buffer containing PMSF (Sigma, St. Louis, MO, USA). A BCA protein detection kit was used to detect the total protein concentration. The same amount of protein was separated by 8–12% sodium alkyl sulfate–polyacrylamide gel electrophoresis and then transferred to the PVDF membrane. The membrane was sealed with a blocking buffer for 2 h at room temperature. Then, the membrane was cultured in blocking buffer at 4 °C overnight with a 1:1000 dilution of primary antibody and washed 3 times with TBST. Finally, the membrane was cultured with a secondary antibody in 1:4000 dilution for 1 h and washed 3 times with TBST. Protein bands were detected using the ECL kit (Yeasen, Shanghai, China) in a chemiluminescence gel imaging system (Tanon, Shanghai, China).

Embryos were frozen in liquid nitrogen and lysed in Tris-SDS buffer (60 mM Tris-HCL, PH6.8, 3.5% SDS). The lysate of each embryos was centrifuged for 7 min at 500g at 4 °C; then we added 2μL Tris-SDS buffer to pellet. Mixtures were heated for 10 min at 100 °C and centrifuged for 5 min at 14,000 rpm at 4 °C. Supernatant was separated by electrophoresis on 8% SDS-PAGE and then the samples were transferred to PVDF membranes (F619537, Sangon, Shanghai, China). Membranes were blocked for 1 h in TBST with 5% non-fat milk powder. The rabbit polyclonal antibody anti-zebrafish Vsx1 was made by Jiaxuan corporation using Vsx1 N terminal 1-144 amino acid as antigen. The Vsx1 primary antibody was diluted 500 times and the HistoneH3 antibody (A2348, ABclonal, Wuhan, China) was diluted 1000 times with 5% milk/TBST solution before used. Membrane was incubated with the diluted Vsx1 antibody and HistoneH3 antibody separately at 4 °C overnight. After being washed for 4 × 10 min with TBST, the member was incubated with secondary antibody (AS014, ABclonal, Wuhan, China) for 1 h at room temperature. Membrane was washed for 4 ×10 min with TBST and stained with ECL kit (36208ES60, Yeasen, Shanghai, China). Band intensity was quantified in ImageJ software and Vsx1 protein was normalized to a corresponding Histone H3 value.