Fig. 8. Ectopic expression of syntaxin 1 altered the distribution and trafficking of TI-VAMP vesicles' cargo CD63. (A) (Upper panels) Non-transfected HeLa cells double stained for endogenous TI-VAMP and CD63. (Lower panels) HeLa cells transfected with syntaxin 1 were fixed and stained for syntaxin 1 and CD63 24 hours after transfection. Note the almost complete colocalization between TI-VAMP and CD63 in non-transfected cells. In cells transfected with syntaxin 1 (asterisk) the CD63 typical punctate staining was lost and instead the nuclear envelope (arrow) was labeled. Insets show confocal images acquired at high magnification of a syntaxin-1-expressing cell. (B) HeLa cells transfected with syntaxin 1 or with syntaxin 1 plus nSec1 were incubated for 1 hour at 4°C with anti-CD63 antibodies prior to fixation and processing for immunofluorescence with anti-syntaxin 1 antibodies. Note the plasma membrane binding of the anti-CD63 antibody in cells co-transfected with syntaxin 1 plus nSec1, which is indistinguishable from non-transfected cells. By contrast, cells expressing and retaining syntaxin 1 in the ER did not bind to anti-CD63 antibodies. Bar, 6 µm. Bar in the inset, 2 µm.