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. 2016 Mar 16;25(10):774–787. doi: 10.1089/scd.2016.0009

FIG. 3.

FIG. 3.

DC secrete full-length CTLA-4 packaged within microvesicular structures. (A) DC-cultured supernatants were precleared with naked protein G-plus beads and subsequently coIP-ed with anti-CD63-coated beads. Depleted supernatants were then analyzed by western blot for flCTLA-4 content. Alternatively, supernatants were treated with various concentrations of NP-40 for 1 h before coIP and then analyzed by western blot for flCTLA-4 remaining in the supernatants. (B, C) Immature and mature DC were analyzed by confocal microscopy to identify Golgi apparatus, Rab5, and CTLA-4 localization. (D) DC-culture supernatants derived from three independent buffy coat products were treated with the Invitrogen Total Exosome Isolation Reagent. Purified EV (30–120 nm) were compared by western blot to remaining supernatant components for CD63, Rab5, IL-12, and CTLA-4. (E) EV purified from DC-cultured supernatants were incubated with anti-CTLA-4-coated beads. The CTLA-4+ pull-down fraction was then compared to the residual fraction by western blot for CTLA-4, CD63, Rab5, and Rab11. EV, extracellular vesicles; flCTLA-4, full-length CTLA-4.