Endocytosed GlyT2 co-localizes with GFP-Rab11, whereas the 4KR mutant fails to internalize and accumulates in the surface.

<p>A-H) MDCK cells were transfected with wild-type GlyT2 and GFP-Rab7 (A, B) or GFP-Rab11 (C, D), or with the 4KR mutant and GFP-Rab7 (E, F) or GFP-Rab11 (G, H). After 48 h the cells were incubated for 30 min with monensin (35 μM) or the vehicle alone and fixed with 4% paraformaldehyde. GFP-Rab7 and GFP-Rab11 were visualized by GFP fluorescence (green), whereas GlyT2 was detected with the anti-GlyT2 antibody (shown in red). Note the increase in intracellular vesicles in monensin-treated cells expressing wild-type GlyT2 (B, D), which colocalizes with GFP-Rab11 (D) but not GFP-Rab7 (B). This increase was not observed in cells expressing the 4KR mutant transporter (F, H). Scale bar = 15 μm. I) Quantification of colocalization using Pearsońs value was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058863#s2" target="_blank">Materials and Methods</a>. Note the increase of colocalization between Rab11 and wild type GlyT2 in the presence of monensin, which cannot be observed with 4KR mutant. The histogram represents the mean ± SEM (n = 3; on average, 30 images per condition were analyzed in each experiment).**, significantly different, p<0.01 by ANOVA with Tukey's post hoc test.