Protein Analysis of Cells and Extracellular Vesicles

Proteins extracted from cells and EVs were examined by Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE). Protein content was estimated according to the Biorad protein assay (BIO-RAD, Hercules, CA, USA), as previously described [17 (link)]. We have analyzed primary antibodies against rabbit polyclonal ANXA1 (1:10,000; Invitrogen; Carlsbad, CA, USA), calreticulin (1:1000; Elabscience; Houston, TX, USA), and mouse monoclonal TSG101 (1:1000; ThermoFisher Scientific; Waltham, MA, USA), CD81 (1:200; Becton Dickinson Labware, Franklin Lakes, NJ, USA), CD63 (1:200; Biolegend; San Diego, CA, USA), and GAPDH (mouse monoclonal, 1:1000; Santa Cruz Biotechnologies, Dallas, TX, USA). The blots were exposed to Las4000 (GE Healthcare Life Sciences; Little Chalfont, UK).