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. 2010 Dec 15;21(24):4338–4348. doi: 10.1091/mbc.E10-05-0407

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© 2010 by The American Society for Cell Biology

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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Figure 1. — Coexpression of channel-active PC2 constructs increases the amount of PC1 at the surface of cultured cells. Unfixed, unpermeabilized cells transiently transfected with PC1 were labeled using an antibody directed against the extracellular FLAG epitope tag appended to the N terminus of PC1 (A, a–d). The composites presented here are compressed stacks of 15 confocal images. Internal PC1 was detected after fixation and permeabilization using an antibody directed against the C-terminal HA epitope on PC1 and the images were merged to show colocalization at the plasma membrane (i–l). There was minimal expression of PC1 on the surface when it was expressed alone (a). These cells, however, expressed PC1 internally, as seen with the antibody directed against the C-terminal HA epitope tag (e). Coexpression of PC2 was associated with the appearance of PC1 protein at the surface (b). PC2-D511V had a substantially smaller effect on PC1 surface localization (c), whereas PC2-L703X caused a distribution of PC1 similar to that seen with PC2 (d). Quantification of five representative compressed stacks showed the extents of the increases in surface PC1 that occurred in the presence of each of the PC2 constructs; asterisk indicates p < 0.05 compared with PC1 alone (B). To address the issue of variable PC1 expression individual cells were imaged and analyzed to determine the immunofluorescence intensity of surface, anti-FLAG antibody (C, top) and internal, anti-HA antibody (C, bottom). For analysis, images were grouped so that images exhibiting similar levels of total PC1 expression could be compared across conditions. Representative images from one such grouping are shown, with the total above-background pixel intensity given in the lower left corner of each image and the ratio of surface to internal immunofluorescence given below the pictures. (C). Averaging the ratios of six such groupings revealed that cells cotransfected with PC2 had a 3.95 ± 0.4-fold increase in surface PC1 compared with those cotransfected with PC2-D511V.