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. 2018 Jan 4;28:151–161. doi: 10.1016/j.ebiom.2018.01.004

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© 2018 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 1 — Hck is required for extracellular vesicle (EV)-associated secretion of ADAM17. (a–b) Hck is present in HIV pEV. (A) Blotting of Hck and indicated tyrosine kinases in pEV purified by differential centrifugation (from 2 ml plasma) from 2 non-viremic HIV patients (HIV.07/0.08) and one healthy control (cont.02). For blotting control the tyrosine kinases were transiently expressed in 293T cells. (b) Blotting of Hck, Yes and Nef in pEV from HIV.08 and a healthy donor (cont.03) isolated by anti-αvβ3-coupled magnetic beads. For blotting control lysates from NIH3T3 and 293T cells transfected with ADAM17 and Nef were used. Beads: bead-isolated pEV; FT: flow through. (c–e) Nef-activated Hck correlates with ADAM17 activation. (c) Blotting of indicated factors in lysates of transiently transfected 293T cells. NAKC: Nef-associated kinase complex (hnRNPK, Lck, PKCδ). (d) Immunoprecipitation of Hck from transiently transfected 293T cells as indicated, and blotting for p-Src. (e) Blotting of indicated factors in lysates of 293T cells stably expressing (stbl. exp.) p59 Hck, and transiently transfected (trans. transf.) as indicated (lower four panels). EV purified from the supernatants were blotted for ADAM17 and Tsg101 (upper two panels). (f–m) EV-associated ADAM17 secretion from myeloid cells requires Hck. (f) Blotting of indicated factors in lysates of immature and mature dendritic cells (iDC/mDC). (g) Blotting of indicated factors in lysates of iDC and mDC, treated/not treated with the tyrosine kinase inhibitor PP2. (h) Blotting of indicated factors in lysates of iDC, mDC, Jurkat and 293T cells, transiently transfected as indicated, or untreated (mDC). (j) Immunoprecipitation of Hck from Nef-transfected iDC and mDC and blotting for indicated factors. (k) Same experiment as shown in (H), blotting EV purified from culture supernatants for indicated factors. (l) Blotting of indicated factors in lysates of Nef-transfected macrophages. (m) Same experiment as shown in (l), blotting EV purified from culture supernatants. Colored boxes in all panels depict results that are described in the text.