VE-2B co-localized with PSD-95 from the TGN to the plasma membrane.

<p>(A) At 10 minutes after release from the ER, VE-2B showed little colocalization with antibody staining to endogenous PSD-95 in the soma (top panels; scale bars are 10 µm). Forty-five minutes after ER release (second row), a time point at which the leading edge of VE-2B reaches the surface, PSD-95 showed significant colocalization with VE-2B. Similar results were obtained with VE-2A (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039585#pone.0039585.s004" target="_blank">Table S1</a>). (B) Colocalization with PSD-95 was also evident in dendrites at 45 minutes and later time points, such as 3 hours (see Fig. 6). VE-2B (top panel) showed co-localization with endogenous PSD-95 (middle panel) in dendrites at 45 minutes after permissive temperature, and later (scale bar 5 µm). (C) Neurons transfected with VE-2B were subjected to 40°C then the medium was exchanged with medium equilibrated at 20°C. Cultures were maintained at 20°C for 1 hour to allow VE-2B cargo exiting the ER to build up in the TGN. Immunostaining for endogenous TGN38 (a TGN marker; top panels) and PSD-95 (second row) in VE-2B transfected neurons subjected to the 20°C temperature manipulation showed robust colocalization (scale bars 5 µm). (D) Quantification of PSD-95 co-localization at different stages of the secretory pathway. There was a significant difference among the groups using a one-way Anova. However, <i>post hoc</i> comparisons demonstrated an insignificant pairwise difference at 10 minutes following ER exit. PSD-95 showed levels of co-localization with VE-2B (open circles) not significantly different from those of VE-2BΔ7 (filled triangles) 10 minutes after ER release. Forty-five minutes after ER exit, VE-2B co-localization with PSD-95 (filled circles) was significantly enhanced at most thresholds when compared to VE-2B colocalization at 10 minutes after ER exit. The significant increase in colocalization of VE-2B with PSD-95 seen at 45 minutes after ER exit could be reproduced by switching media to 20°C for 1 hour (open squares). To verify that VE-2B was concentrated in the TGN after 1 hour 20°C incubation, colocalization of VE-2B with TGN38 was quantified (filled squares).