FIGURE 1.

Knockdown of syntaxin 4 results in inhibition of basolateral trafficking. A, Western blot analysis of syntaxin 4 expression levels in cells expressing two different hairpins to syntaxin 4 (KD2 and KD3). Two clones for each hairpin are shown. A cell line expressing a hairpin to GFP (GFPKD) was used as a control cell line. Actin is the loading control. B–F, MDCK cell line depleted in syntaxin 4 (syn4KD3) and a control cell line (GFPKD) were grown for 5 days on filter, fixed, and stained. B, gp135/podocalyxin (magenta) and β-catenin (green) (z-scan). C, cells stained for several different basolateral proteins (syntaxin 4, Na,K-ATPase β1, E-cadherin, β1-integrin, and Scribble). D, cells stained for tight junction proteins (occludin, ZO-1, claudin 2, and sec6). Staining for actin is also shown. Images represent projections of several x-y scans. Representative single x-y scans are shown under supplemental Fig. S1B. E, cells stained for laminin (top, projections of x-y scans) and laminin (green) and actin (magenta) (bottom, z-scans). F, cells stained for SNAP23. Scale bars, 20 μm.