Exoab cd9a 1

Exosomes were lysed in cold RIPA buffer with PMSF. The total protein concentration was determined by BCA assay. Protein samples were mixed with 5× loading buffer and separated by 10% SDS polyacrylamide gels. Then the proteins were transferred to PVDF membranes (Millipore, NJ, United States) and incubated with 5% non-fat milk at room temperature for 1 h. Exosomes were identified using 2 positive markers including CD9 (1:1,000; EXOAB-CD9A-1, System Biosciences) and CD63 (1:1,000; EXOAB-CD63A-1, System Biosciences). Calnexin (1:1,000; YT0613, Immunoway Biotechnology) was used as a negative marker for exosomes.

Equal amounts (5 μg) of EVs, CEM (T lymphoblast) cell lysate, and two ExoQuick EV-depleted supernatant samples were analyzed using SDS-PAGE followed by immunoblotting with antibodies against known EV protein markers Flotillin1 (clone EPR6041; Abcam), CD9 (EXOAB-CD9A-1; System Biosciences), and the EV purity markers GM130 (ab52649; Abcam) and Apolipoprotein A1 (ab64308; Abcam). Full immunoblots are in Supp. Fig. 2.

To obtain protein lysate cells were resuspended in RIPA buffer and exosomes in 8 M Urea + 2.5% SDS. The lysates were then separated by SDS-PAGE and subjected to western blotting. Western blot analysis was performed according to standard procedures using polyvinylidene difluoride membranes and an enhanced chemiluminescence system (GE Healthcare). The following antibodies were used: CD63 (EXOAB-CD63A-1, System Biosciences, 1:1000), CD9 (EXOAB-CD9A-1, System Biosciences, 1:500), Alix (#2171, Cell Signaling, 1:500), and TSG101 (EXOAB-TSG101-1, System Biosciences, 1:500). To test plasma samples reactivity against exosome lysate, blotted membranes were incubated with plasmas at a 1:100 dilution overnight at 4 °C and then with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (1 h, 1:4000; GE Healthcare).

Protein quantification was performed using Pierce BCA Protein Assay (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's recommended Microplate assay procedure. Absorbance was measured with a SpectraMax M5 multi-mode microplate reader using SoftMax Pro data acquisition and analysis software (MolecularDevices, Sunnyvale CA). Vesicle isolates were denatured in 4x Laemmli sample buffer at 100°C for 10 minutes. Proteins were separated using 4-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis in Tris/Glycine/SDS running buffer and transferred to Immun-Blot PVDF membrane (all reagents and supplies from Bio-Rad, Hercules, CA). Immunoblotting was performed with the following primary antibodies: CD9 (EXOAB-CD9A-1, System Biosciences, Palo Alto, CA); TSG101 (ab125011, Abcam, Cambridge, MA); and TPM3 (H00007170-M02, Novus Biologicals, Littleton, CO). Blots were washed and incubated with appropriate HRP-conjugated secondary antibodies (Amersham ECL, GE, Uppsala, Sweden) and detected using Pierce ECL western blotting substrate (Thermo Fisher Scientific) with chemiluminescence-optimized autoradiography film. Densitometry analysis was performed on digitized images using NIH ImageJ image processing and analysis software.

Protein quantification was performed using Pierce BCA (bicinchoninic acid) protein assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommended microplate assay procedure. Absorbance was measured with a SpectraMax M5 multi-mode microplate reader using SoftMax Pro data acquisition and analysis software (MolecularDevices, San Jose, CA, USA). Vesicle isolates were denatured in 4× Laemmli sample buffer at 100 °C for 10 min. Proteins were separated using 4–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis in Tris/Glycine/SDS running buffer and transferred to Immun-Blot PVDF (polyvinylidene difluoride) membrane (all reagents and supplies from Bio-Rad, Hercules, CA, USA). Immunoblotting was performed with the following primary antibodies: CD9 (EXOAB-CD9A-1, System Biosciences, Palo Alto, CA, USA); TSG101 (EXOAB-TSG101-1, System Biosciences). Blots were washed and incubated with appropriate HRP-conjugated secondary antibodies (Amersham ECL, Cytiva, Marlborough, MA, USA) and detected using Pierce ECL western blotting substrate (Thermo Fisher Scientific) with chemiluminescence optimized autoradiography film.