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. 2012 Apr 3;10(4):e1001299. doi: 10.1371/journal.pbio.1001299

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Nagawa et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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PIN1-GFP was transiently expressed or coexpressed with DN-ROP2 and/or ANTH(C) constructs in tobacco PCs for 3 d and visualized by confocal microscopy. DN-ROP2 and/or ANTH(C) expression was induced by DEX treatment for 6 h before observation. Ika (5 µM) treatment was carried out by infiltration 2 h before observation. (A) Internalization of PIN1-GFP into vesicles (arrowheads) was observed in control (upper left) and was greatly enhanced by DN-ROP2 expression (lower left). The formation of these PIN1-GFP vesicles was inhibited by the expression of ANTH(C) (middle panels) or treatment with Ika (right panels) in both control (upper panels) and DN-ROP2 cells (lower panels). (B) Quantitative analysis of vesicle numbers shown in (A). Medial sections of confocal scanning images were taken from ten different leaves in at least three independent experiments, and the mean number of vesicles per area of cells was determined. Error bars represent SD (n = 10). p-Values were determined by two-tailed Student's t test assuming equal variances (***, p<0.001).