Anti-Fas Antibody, clone 7C10

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	-Mouse (1:500)

Protocol tips
-Mouse (1:500)

Publication protocol

Cells and purified exosomes were lysed in M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) with protease and phosphatase inhibitors (Roche), and proteins were quantified using a protein concentration assay (Bio-Rad Laboratories). For Western blotting of cell lysis, 20 μg of proteins was separated by SDS–polyacrylamide gel electrophoresis and transferred to 0.2-μm nitrocellulose membranes (Millipore). For Western blotting analysis of exosome, exosome protein was isolated from the culture supernatant of 4 × 106 cells, and 10 μg of total exosome proteins was loaded. The membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 for 1 hour, followed by incubation overnight with the primary antibodies diluted in blocking solution according to the manufacturer’s instructions. Antibodies to mouse Fas (05–351) were purchased from Millipore. Antibodies to mouse Fap-1 (sc-15356), Cav-1 (sc-894), and SNAP25 (sc-7538) were purchased from Santa Cruz Biotechnology. Antibodies to mouse IL-1RA (ab124962) and VAMP5 (ab85581) were purchased from Abcam. Antibody to mouse β-actin (A5441) was purchased from Sigma-Aldrich. The membranes were then incubated under room temperature for 1 hour in species-related horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology) diluted at 1:10,000 in blocking solution. Immunoreactive proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and Biomax film (Kodak); the sensitivity of this substrate can be used to detect low-picogram amounts of protein in polyvinylidene difluoride membrane. The relative density was measured using ImageJ 1.49v software (Wayne Rasband). The quantification of Western blotting for total EV protein was normalized against the control group or GMSC group, and the quantification of the other Western blotting experiments was normalized against loading control β-actin.

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