Activation induces up-regulation of VAMP8 expression and polarization of VAMP8 to immune synapses in human CD8⁺ T cells. (A) Relative expression of VAMP8 transcripts in unstimulated or bead-stimulated (stim.) CD8⁺ T cells was determined by qRT-PCR. UBC (encoding ubiquitin C) transcripts were used for normalization. Graph depicts data from five individual donors (paired t test, **, P = 0.0035). (B) VAMP8 expression in naive, bead-stimulated for the indicated days, or SEA-stimulated for 5 d CD8⁺ T cells was determined by Western blot analysis. (C) Expression of VAMP8 relative to GAPDH in CD8⁺ T cells after various stimulations, as indicated. Graphs represent mean, normalized expression of VAMP8 in five individuals with SD indicated (paired t test, *, P < 0.05; **, P < 0.005; ***, P < 0.001). (D) SIM images of endogenous VAMP8 localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. (E) SIM images of ectopically expressed VAMP8-TFP localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. Bars, 2.5 µm.