Subcellular distribution of native P2X6 subunits in hippocampal neurons <i>in vitro</i>.

<p>(<b>a,b</b>) Immunofluorescence images of 3(<b>a</b>) and 18(<b>b</b>) days-old primary cultures of hippocampal neurons labeled with antibodies against P2X6 and β-III Tubulin. Nuclei were counterstained with DAPI. Confocal image and orthogonal views of hippocampal neurons show that P2X6 immunostaining is localized in ER compatible location and inside the nucleus, and shows a more dotted pattern as days in culture passed. Scale bar 5 μm. (<b>c-e</b>) Immunofluorescence images 16–18 days-old primary cultures of hippocampal neurons labeled with antibodies against P2X6 and NeuN (<b>c</b>), fibrillarin (<b>d</b>) or SC35 (<b>e</b>). Nuclei were counterstained with DAPI. Confocal image and orthogonal views of hippocampal neurons show that P2X6 immunostaining is localized in ER compatible location and inside the nucleus without co-localization with NeuN (<b>c</b>), fibrillarin (<b>d</b>) or SC35 (<b>e</b>). Scale bar 5 μm. (<b>f</b>) Western blot of protein extracts from neuroblastoma cells transfected with either P2X6-YFP chimeric protein or YFP vector and labelled with antibodies against GFP and P2X6. (<b>g, h</b>) Immunofluorescence of hippocampal neurons transfected with either empty vector YFP (<b>g</b>) or P2X6-YFP (<b>h</b>). Confocal image and orthogonal views in (<b>h</b>) shows that P2X6 and YFP staining are distributed in cytosol, but are predominantly accumulated close to the nuclear membrane and also inside the nucleus without co-localization with DAPI. Scale bar 5 μm. (<b>i</b>) P2X6 expression was knocked down in neuroblastoma cells transfected with P2X6-YFP using three specific shRNAs. shRNA against luciferase (shRNA Luc) was used as negative control. Protein extracts were immunoblotted with anti-GFP antibody in order to characterize the efficiency of each shRNA. (mean±s.e.m, n = 3 independent experiments, * P<0.05, ** P<0.01 unpaired Student's t-test). (<b>J</b>) Endogenous P2X6 expression was knocked down in hippocampal neurons transfected with shRNA 1, showing less nuclear inclusions than non-transfected neurons. Scale Bar 10 μm. Bar graph represents the number of nuclear inclusions detected with anti-P2X6 antibody in neuronal nucleus (mean±s.e.m., n = 30 cells *P<0.01 unpaired Student’s t-test). (<b>k, l</b>) Cartoon of intraventricular electroporation of E14.5 embryo mice (<b>k</b>). Confocal microphotography of P0 mice brain from both YFP or P2X6-YFP electroporated mice showing the nuclear and perinuclear location of P2X6 in P2X6-YFP transfected mice (<b>l</b>). V, ventricle. Scale Bar 50 μm.