Emmprin

Cells and exosomes were lysed in a lysis buffer (1% Triton-x100, 1 mM EDTA, 150 mM NaCl, 20 mM Tris pH 7.5) supplemented with 1 × cOmplete™ EDTA-free Protease Inhibitor Cocktail (11836170001, Roche (Basel, Switzerland)) and protein concentrations were determined using a Bradford assay (Bio-Rad) in relation to a BSA standard curve. Proteins were separated according to molecular weight by SDS-PAGE and transferred onto a PVDF membrane (Scientific Lab Supplies 10600023). For protein detection, membranes were blocked with 5% nonfat milk and 1% Tween/PBS, and subsequently incubated with primary antibodies against MMP-2 (Abcam, 86607, 1:500), EMMPRIN (Santa Cruz, 46700, 1:500), CD9 (Cell signalling, 13174, 1:1000), Alix (Cell signalling, 2171, 1:1000), Annexin V (Cell signalling, 8555, 1:1000) and Histone 4 (Cell signalling, 41328, 1:1000). Appropriate horseradish peroxidase-conjugated secondary antibodies were applied. GAPDH (Sigma-Aldrich, 406609, 1:2000) was used as the loading control. Protein signals were detected using an enhanced chemiluminescence (ECL) solution (Thermo Fisher Scientific (Waltham, MA, USA); 32106). Chemiluminescence was then measured in the LAS-3000 mini biomolecular imager.

Antibodies for myostatin and MMP‐9 were purchased from Abcam (Cambridge, MA), EMMPRIN and iNOS antibodies and all secondary antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The glyceraldehyde 3‐phosphate dehydrogenase (GAPDH ) antibody was purchased from EMD Millipore (Billerica, MA). All PCR primers were purchased from Invitrogen (Carlsbad, CA).