Supplemental Material

This article contains the following supporting material:

• Supplementary Figure 1 - Effect of fixation conditions on the distribution of exocyst subunits in polarized MDCK cells. Cell were fixed using methanol (A&D), fixed using a pH-shift protocol (B&E), or permeabilized with saponin for 5 min at 4°C prior to fixation using the pH-shift protocol (C&F). (A-C) Distribution of Sec6 (detected using the rSec6 monoclonal antibody; green) and ZO-1 (red). (D-F) Distribution of Sec8 (detected using the rSec8 monoclonal antibody; green) and ZO-1 (red). (A-F) XZ sections through the monolayer are shown at the top of each set and single merged optical sections from the apical pole of the cell or at the level of the tight junctions are shown below. Scale bar = 10 µm.
• Supplementary Figure 2 - Distribution of exocyst subunits in MDCK cells. (A) Cells were permeabilized with saponin for 5 min, fixed using a pH shift protocol, and then stained with the rSec6 monoclonal antibody (green) and an antibody to ZO-1 (red). The top two panels are identical to those shown in Supplementary Figure 1C. (B) Cells were permeabilized with saponin for 5 min, fixed using a pH-shift protocol, and then stained with the Sec8 monoclonal antibody 10C2 (green) and an antibody to ZO-1 (red). (C) Cells were fixed using a pH-shift protocol and then stained with the 3F3 antibody (green) and an antibody to ZO-1 (red). (A-C) XZ sections are shown at the top of each column, and individual merged optical sections at the designated levels are shown below. Scale bar = 10 µm.
• Supplementary Text