Anti hsp70

Ten µL aliquots of either a UC preparation or individual SEC fractions were analyzed by Western blot against human CD71. Membranes were probed with rabbit polyclonal anti-CD71 (ab84036) at 1:250 dilution for 1 hour at RT. A goat anti-rabbit IgG coupled to HRP (Sigma, A6154) was used at a dilution of 1:2,500 for 1 hour at RT. Revealing was performed using ECL Western Blotting Substrate (Pierce™) in ImageQuant LAS 4000 (GE Healthcare Life Sciences). Additionally, 20 µL aliquots of UC preparations were analyzed to confirm the presence of HSP70, GAPDH and stomatin in HuRex. Membranes were incubated for 1 hour at RT with primary antibodies anti-HSP70 (Santa Cruz Biotechnology, W27 sc-24) at 1:250 dilution, anti-GAPDH (Sigma, G9545) at 1:500 dilution or anti-stomatin (Invitrogen, PA5-30019) at 1:250 dilution. Subsequently, membranes were washed and incubated for 1 h at RT with the Li-Cor IRDye-labeled secondary antibodies IRDye® 800CW goat-anti-mouse (925-32210, Li-Cor Biosciences) at 1:15,000 dilution or IRDye® 680RD goat anti-rabbit (925-68021, Li-Cor Biosciences) at 1:20,000 dilution. Blots were analyzed with the Odyssey near-infrared system (Li-Cor Biosciences) having the intensity of 700 channel set up at 5 and the one of 800 channel at 7. Images were edited using the software Image J (NIH).

Five micrograms of EVs A were incubated with 4-μm diameter aldehyde/sulfate latex beads (Invitrogen, CA, USA), as detailed in Ref. (25 (link), 26 (link)). Bound EVs were spun down, and the unoccupied sites were saturated with 100 mM glycine. Then, the EVs were incubated with anti-mouse CD24-PE, anti-CD8-PE, and isotype-matched controls (eBioscience, CA, USA). After incubation with anti-MHC I-Biotin, CD9-Biotin, CD81-Biotin (eBioscience, CA, USA), anti-HSP-70, and anti-HSP-90 (Santa Cruz Biotechnology, TX, USA) primary antibodies, the EVs-coated beads were incubated with a streptavidin–PE-conjugated (Invitrogen, CA, USA) or an Alexa647-conjugated secondary antibody, respectively (Jackson Immuno Research, PA, USA). Fluorescence was measured in a BD FACSCalibur flow cytometer (BD Biosciences, CA, USA). The data were analyzed with the Flowing 2.5.1 software.

Ten micrograms of EVs or cell lysates were resuspended in Laemmli sample buffer and separated by electrophoresis on 10% SDS-polyacrylamide gels. Gels were electroblotted onto nitrocellulose paper (Hybond-ECL nitrocellulose membrane, 0.2-μm transfer membrane; GE Amersham Life Sciences, USA) and blocked overnight with 5% non-fat dry milk in Tris buffer saline (B-TBS). Blots were incubated overnight at 4°C with mouse anti-CD63, anti-TSG-101, anti-ALIX, anti-HSP-70, or anti-HSP-90 antibodies (Santa Cruz Biotechnology, TX, USA). Finally, blots were washed and incubated with an anti-mouse HRP-conjugated or an anti-rabbit HRP-conjugated serum. Membranes were revealed with the ECL kit (GE, Amersham Life Sciences, USA) following the manufacturer’s instructions. The GE Healthcare, Image Quant TM-RT ECL, Version 1.0 software was used to detect specific proteins (25 (link)).