Anti hsp70

Total proteins from the THP-1 cells treated with MM1.S- and SW480-derived SEVs and from MM1.S- and SW480-derived SEVs were isolated and analyzed by SDS-PAGE followed by Western Blotting. The primary antibodies used in the experiments were as follows: anti-PD-L1 antibody (Abcam, Cambridge, UK, ab213524, diluition 1:1000; negative and positive controls of the antibody are included in Supplementary Figure S5), anti-pSTAT3 (R&D System, AF4607-SP, diluition 1:500), anti-STAT3 (Novus Biologicals, Denver, CO, USA, NBP2-24463, diluition 1:1000), anti-HSP70 (Novus Biologicals, NB600-1469, diluition 1:1000), anti-HSC70 (Santa Cruz, Dallas, TX, USA, sc-7298, diluition 1:500), anti-Calnexin (Santa Cruz, sc-23954, diluition 1:500), anti-Tubulin (Santa Cruz, sc-398103, diluition 1:1000), anti-β Actin (Santa Cruz, sc-81178, diluition 1:1000), anti-GAPDH (Santa Cruz, sc-47724, diluition 1:1000), and anti-NF-kB (Novus, NB100-2176, diluition 1:500). After overnight incubation with the primary antibodies at 4 °C, the membranes were incubated with HRP-conjugated secondary antibody (Thermo Fisher Scientific, Cambridge, MA, USA) for 1 h at 4 °C; the chemiluminescent signal was detected by Chemidoc (Biorad, Milan, Italy).

Purified exosomes (200 μg) were incubated with 10 μL of 4-μm-diameter aldehyde/sulfate latex beads (Invitrogen, Eugene, OR) in PBS for 1 h at room temperature with gentle agitation. After washing with PBS, samples were blocked with 200 mM glycine for 30 min and washed again. Beads were incubated with the following exosome-associated antibodies for 30 min: anti–CD63 (Millipore, Billerica, MA, USA), anti-CD9 (BD Biosciences, San Jose, CA, USA), anti-HLA-A,B,C (BioLegend, San Diego, CA, USA), anti-TSG101 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HSP70 (Novus Biologicals, Littleton, CO, USA), anti-lysosomal membrane glycoproteins CD107a (LAMP-1) (Miltenyi Biotec, Auburn, CA, USA), anti-MICA/B (eBioscience, Grand Island, NY, USA), anti-CD81 (ProSci, Fort Collins, CO, USA), anti-TGF-β (Peprotech, Rocky Hill, NJ, USA), and anti-FasL (Boster, Pleasanton, CA, USA). Staining with secondary antibodies for 30 min followed, using goat anti-rabbit Alexa Fluor 488 (Invitrogen, Eugene, OR, USA) and goat anti-mouse FITC (Becton Dickinson, Franklin Lakes, NJ, USA). A “bead-only” control as well as isotype-matched antibody controls were also prepared. Samples were washed twice, fixed with 1% paraformaldehyde and analyzed using a MACSQuant Analyzer and FlowJo software. Single beads were gated for fluorescence analysis.