Figure 4.

In nonpolarized MDCK cells, apical t-SNAREs localize to the VAC, whereas basolateral SNAREs are excluded from it. MDCK cells stably expressing syntaxins 2, 3, 4, and 11 and SNAP-23 were seeded onto glass coverslips in regular medium. After 2 h, the cells were switched to LCM and incubated for 16 h. Cells were fixed, permeabilized, and stained for the individual SNAREs (left column) and with an antibody against an endogenous apical plasma membrane protein, gp135 (right column). gp135 localizes to the VAC. Syntaxins 2, 3, and 11 and SNAP-23 colocalize with gp135 in VACs. In contrast, syntaxin 4 is excluded from VACs (arrows indicate VACs for better orientation). Syntaxins 2 and 11 and SNAP-23 are also found in large intracellular compartments that exclude gp135 (arrows) in addition to gp135-positive VACs. Bars, 5 μm.