, 2010). To test whether PrP and α2δ-1 interacted physically, we immunoprecipitated PrP from cerebellar extracts of Tg(WT) and Tg(PG14) mice, and immunoblotted the precipitated fractions with an antibody raised against the α2 polypeptide of α2δ-1. As shown in Figure 5A, an immunoreactive band of ∼145 kDa was detected in immunoprecipitates of Tg(WT) and Tg(PG14)

but not in Prnp0/0 mice, or when the immunoprecipitation was done in the absence of the anti-PrP antibody. After deglycosylation with PNGaseF, this band shifted to an apparent molecular buy 3-Methyladenine weight of 107 kDa, as expected for the α2 polypeptide ( Figure S5A) ( Davies et al., 2006). The interaction was confirmed in the reverse experiment in which α2δ-1 was immunoprecipitated http://www.selleckchem.com/products/Adriamycin.html from cerebellar extracts and PrP detected by immunoblot (Figure 5B), and was also seen in primary cultured CGNs (Figure S5B) and transiently transfected HeLa cells (Figure S5C). HC-deleted PrP molecules coimmunoprecipitated with

α2δ-1 (Figure S5C), indicating that PrP region 114–121 was not essential for the interaction. Next, we tested whether the distribution of α2δ-1 was altered in cells expressing PG14 PrP. HeLa cells were cotransfected with plasmids encoding the CaVα1A, CaVβ4, and α2δ-1 subunits, and either wild-type or PG14 PrP-EGFP fusion proteins, and analyzed by confocal microscopy after immunofluorescent staining of α2δ-1. Consistent with previous localization of nonfluorescent and EGFP-fused PrPs (Biasini et al., 2010, Fioriti et al., PDK4 2005 and Ivanova et al., 2001), the majority of wild-type PrP localized on the cell surface (Figures 6A and 6J), whereas PG14 PrP was mostly found in intracellular

compartments (Figures 6D and 6J). In cells expressing wild-type PrP, α2δ-1 was efficiently expressed on the plasma membrane where it colocalized with PrP (Figures 6B, 6C, and 6K). In contrast, α2δ-1 was weakly expressed on the surface of PG14 PrP-expressing cells, and was mostly found in perinuclear patches where it colocalized with PrP (Figures 6E, 6F, and 6K), and with ER and Golgi markers (data not shown). This was seen in cells with high or low expression levels, ruling out that the abnormal localization of α2δ-1 was due to overexpression. The CaVα1A pore-forming subunit also accumulated intracellularly in PG14 PrP-expressing cells (Figures S6A–S6H), whereas there was no effect on the localization of 5′ nucleotidase (5′NT), a raft-resident GPI-anchored protein that does not belong to the VGCC complex (Davies et al., 2010) (Figures S6I–S6P). In cells expressing PG14/ΔHC PrP, α2δ-1 was more efficiently delivered to the cell surface, indicating that intracellular retention of mutant PrP played a role in the trafficking defect (Figures 6H, 6I, and 6K).