Western blot detection system

The extracellular vesicles were submitted to lysis using the RIPA buffer added with protease inhibitor cocktail. The resultant was analysed for total proteins using the protein estimation kit (Sigma Aldrich USA, Catlog# 51254). The proteins were incubated with specific antibodies overnight for obtaining blots. The antibodies used were specific for IL-6 (1:500) (ab6672), IL-1β (1:500) (ab9722), TNF-α (1:500) (ab6671), CD81 (1:1000) (ab109201), CD63 (1:1000) (ab231975), CD9 (1:1000) (ab223052) and HSP70 (1:1000) (ab2787), all the antibodies were obtained from Abcam USA. The blots were detected using Western blot detection system (Thermo Fisher USA).

Fluorescein-cadaverine (FC, Molecular Probes^TM A10466) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Putrescin, 4’,6-diamidino-2-phenylindole (DAPI), TGase assay kit (CS1070-1KT), RPMI 1640 medium, and goat serum were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Diethyl aminoethyl (DEAE) and Heparin Sepharose were purchased from Cytiva (Marlborough, MA, USA) and Sigma-Aldrich, respectively, and nitrocellulose membrane (Amersham Protran Supported) was purchased from GE Healthcare (Chicago, IL, USA). Human TGase 2 rabbit polyclonal antibodies (orb2986) were purchased from Biorbyt (Cambridge, UK). Horseradish-peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were purchased from Novus Biologicals Europe, UK. FITC-tagged anti-rabbit antibodies were obtained from Chemicon (Temecula, CA, USA). Fetal bovine serum (FBS, South America origin, EU Approved) was obtained from EuroClone, Italy. Protease inhibitor cocktail and the Western Blot detection system SuperSignal™ West Femto Maximum Sensitivity Substrate were obtained from Thermo Scientific (Waltham, MA, USA). Protein molecular mass markers ECL Plex Fluorescent Rainbow and Precision Plus Protein™ Dual Colour Standards were obtained from BIO-RAD (Hercules, CA, USA). All other chemicals were obtained from Sigma-Aldrich.