p2x6  (Alomone Labs)

Journal: PLoS ONE

Article Title: Age-Related Nuclear Translocation of P2X6 Subunit Modifies Splicing Activity Interacting with Splicing Factor 3A1

doi: 10.1371/journal.pone.0123121

Figure Lengend Snippet: Extracellular region of P2X6 is responsible for nuclear location. ( a ) Western blot of protein extracts from neuroblastoma cells transfected with constructions containing YFP as control, P2X6 and N-terminal defective P2X6 fused with YFP (N14P2X6-YPF). ( b ) Confocal images and orthogonal views of N2a neuroblastoma cells transfected with YFP, P2X6-YFP and N14P2X6-YPF. Subcellular distribution of both YFP and N14P2X6-YFP were exclusively cytosolic, whereas P2X6-YFP shows cytoplasmic and nuclear location. Scale Bar 10 μm. ( c ) Western blots of protein extracts from neuroblastoma cells transfected with constructions containing different P2X6 subregions fused with c-myc epitope and labelled with anti-myc antibody. ( d ) Confocal images and orthogonal views of N2a neuroblastoma cells transfected with P2X6-myc, P2X6 extracellular region (EXT-P2X6-myc), and second transmembrane domain (TM-P2X6-myc). Subcellular distribution of TM-P2X6-myc were exclusively cytosolic, whereas EXT-P2X6-myc shows cytoplasmic and nuclear location. Scale bar 10 μm. ( e ) Neuroblastoma cells transfected with GCL-myc and P2X6 extracellular domain fused with GCL-myc (EXT-P2X6-GCL-myc) constructions show different locations. Subcellular distribution GCL-myc were exclusively cytosolic, whereas EXT-P2X6-GCL-myc shows cytoplasmic and nuclear location. Scale bar 10 μM. ( f ) Protein alignment of human P2X subunits using ClustalX algorithm and sequence analysis by PFAM database of protein families revealed the presence of WD40 domain exclusively in P2X6 subunit.

Article Snippet: Cells were then incubated with primary antibodies at the following dilutions: rabbit anti-P2X6 (Alomone Labs, 1:200), mouse anti-c-myc (Life Technologies, 1/1000), mouse anti-α-tubulin (Sigma Aldrich, 1/1000), mouse anti-Map-2 (Sigma Aldrich, 1/500), mouse anti-NeuN (Chemicon, 1/500), mouse anti SC35 (Sigma Aldrich, 1/1000), mouse anti fibrillarin (Santa Cruz, 1/200),rabbit anti-GFP (Life Technologies, 1/500) and mouse anti β-III-tubulin (Promega, 1/1000), for 1 h at room temperature.

Techniques: Western Blot, Transfection, Sequencing

Journal: PLoS ONE

Article Title: Age-Related Nuclear Translocation of P2X6 Subunit Modifies Splicing Activity Interacting with Splicing Factor 3A1

doi: 10.1371/journal.pone.0123121

Figure Lengend Snippet: Age-dependent expression of P2X6 is not related with senescence. ( a ) Western blot analysis for expression of P2X2, P2X4 and P2X6 proteins in mice (from 1 to 18 month-old) ( b ) Graph represent P2X6, P2X2 and P2X4 levels relative to 1 month-old mice. Levels for P2X6 expression is on average 2.58±0.14-fold higher, P2X4 subunit only increases 1.45±0.19-fold at 8 month, and P2X2 level remains invariable (mean±s.e.m, n = 7 mice, * P

Article Snippet: Cells were then incubated with primary antibodies at the following dilutions: rabbit anti-P2X6 (Alomone Labs, 1:200), mouse anti-c-myc (Life Technologies, 1/1000), mouse anti-α-tubulin (Sigma Aldrich, 1/1000), mouse anti-Map-2 (Sigma Aldrich, 1/500), mouse anti-NeuN (Chemicon, 1/500), mouse anti SC35 (Sigma Aldrich, 1/1000), mouse anti fibrillarin (Santa Cruz, 1/200),rabbit anti-GFP (Life Technologies, 1/500) and mouse anti β-III-tubulin (Promega, 1/1000), for 1 h at room temperature.

Techniques: Expressing, Western Blot, Mouse Assay

Journal: PLoS ONE

Article Title: Age-Related Nuclear Translocation of P2X6 Subunit Modifies Splicing Activity Interacting with Splicing Factor 3A1

doi: 10.1371/journal.pone.0123121

Figure Lengend Snippet: Subcellular distribution of native P2X6 subunits in hippocampal neurons in vitro . ( a,b ) Immunofluorescence images of 3( a ) and 18( 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. ( c-e ) Immunofluorescence images 16–18 days-old primary cultures of hippocampal neurons labeled with antibodies against P2X6 and NeuN ( c ), fibrillarin ( d ) or SC35 ( e ). 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 ( c ), fibrillarin ( d ) or SC35 ( e ). Scale bar 5 μm. ( f ) 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. ( g, h ) Immunofluorescence of hippocampal neurons transfected with either empty vector YFP ( g ) or P2X6-YFP ( h ). Confocal image and orthogonal views in ( h ) 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. ( i ) 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

Article Snippet: Cells were then incubated with primary antibodies at the following dilutions: rabbit anti-P2X6 (Alomone Labs, 1:200), mouse anti-c-myc (Life Technologies, 1/1000), mouse anti-α-tubulin (Sigma Aldrich, 1/1000), mouse anti-Map-2 (Sigma Aldrich, 1/500), mouse anti-NeuN (Chemicon, 1/500), mouse anti SC35 (Sigma Aldrich, 1/1000), mouse anti fibrillarin (Santa Cruz, 1/200),rabbit anti-GFP (Life Technologies, 1/500) and mouse anti β-III-tubulin (Promega, 1/1000), for 1 h at room temperature.

Techniques: In Vitro, Immunofluorescence, Labeling, Immunostaining, Western Blot, Transfection, Plasmid Preparation, Staining, Expressing, shRNA, Luciferase, Negative Control

Journal: PLoS ONE

Article Title: Age-Related Nuclear Translocation of P2X6 Subunit Modifies Splicing Activity Interacting with Splicing Factor 3A1

doi: 10.1371/journal.pone.0123121

Figure Lengend Snippet: Expression of P2X6 subunit in adult mice hippocampus. ( a-c ) Immunohistochemical analysis of hippocampus sections from 8 month-old mice. Cells were labeled with antibodies against P2X6 ( a ), P2X4 ( b ) and P2X2 ( c ) subunits. Insets depict enlarged views (40X magnification) of CA3 hippocampal region showing a dotted pattern for P2X6 into the nucleus in addition to cytoplasmic staining ( a ), somatodendritic stainining for P2X4 ( b ) and axonal and cytoplasmic staining for P2X2( c ). Scale bar 200 μm. ( d-f ) Immuno-electron microscopy analysis of a CA3 hippocampal neuron from 8 months-old wild type mouse labeled with anti-P2X6 antibody. The staining is observed both in the cytoplasm (cyt, white arrows) and into the nucleus (nuc, white asterisks). Scale bar 2 μm. ( d-e ) Inset of the area indicated in ( e ) showing a 3.5 X magnification of this indicated area. P2X6 positive regions are mainly located in patches added to the inner nuclear membrane ( e ).

Article Snippet: Cells were then incubated with primary antibodies at the following dilutions: rabbit anti-P2X6 (Alomone Labs, 1:200), mouse anti-c-myc (Life Technologies, 1/1000), mouse anti-α-tubulin (Sigma Aldrich, 1/1000), mouse anti-Map-2 (Sigma Aldrich, 1/500), mouse anti-NeuN (Chemicon, 1/500), mouse anti SC35 (Sigma Aldrich, 1/1000), mouse anti fibrillarin (Santa Cruz, 1/200),rabbit anti-GFP (Life Technologies, 1/500) and mouse anti β-III-tubulin (Promega, 1/1000), for 1 h at room temperature.

Techniques: Expressing, Mouse Assay, Immunohistochemistry, Labeling, Staining, Immuno-Electron Microscopy

Journal: PLoS ONE

Article Title: Age-Related Nuclear Translocation of P2X6 Subunit Modifies Splicing Activity Interacting with Splicing Factor 3A1

doi: 10.1371/journal.pone.0123121

Figure Lengend Snippet: P2X6 subunit interacts with SF3A1 and Spectrin α2 in nuclear extracts from hippocampus of 8 month-old mice and alters splicing efficiency in vitro . ( a ) Western blot of subcellular fractions from 8 month-old mice hippocampus. P2X6 subunit was located in both the cytosolic (cyt) and the nuclear fraction (nuc), as identified by localization with either glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or NeuN (neuronal nuclei marker)/histone 2b (H2b), respectively. ( b ) Nuclear extracts immunoprecipitated with anti-P2X6 antibody were resolved in a 2D electrophoresis gel and selected spots were isolated, processed and identified by MALDI/TOF (n = 3 mice). ( c-f ) Nuclear extracts immunoprecipitated with either IgG or anti-P2X6 antibodies were analysed by immunoblotting with antibodies against SF3A1 ( c ) and spectrin α-2( d ). Nuclear extracts were also immunoprecipitated with spectrin α-2 ( e ) or SF3A1 ( f ) antibodies and immunoblotted with anti-P2X6 antibody. ( g ) Scheme shows four DNA constructions prepared for splicing activity quantitation and their respective protein translations. ( h ) Splicing inhibitor isoginkgetin (IGK) was used to validate the molecular tools employed to quantify the splicing efficiency. As expected TPI I firefly luciferase-dependent luminescence decreases and TPI II firefly luciferase-dependent luminescence increases when splicing is impaired (mean±s.e.m., n = 5 independent experiments, * P

Article Snippet: Cells were then incubated with primary antibodies at the following dilutions: rabbit anti-P2X6 (Alomone Labs, 1:200), mouse anti-c-myc (Life Technologies, 1/1000), mouse anti-α-tubulin (Sigma Aldrich, 1/1000), mouse anti-Map-2 (Sigma Aldrich, 1/500), mouse anti-NeuN (Chemicon, 1/500), mouse anti SC35 (Sigma Aldrich, 1/1000), mouse anti fibrillarin (Santa Cruz, 1/200),rabbit anti-GFP (Life Technologies, 1/500) and mouse anti β-III-tubulin (Promega, 1/1000), for 1 h at room temperature.

Techniques: Mouse Assay, In Vitro, Western Blot, Marker, Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Isolation, Activity Assay, Quantitation Assay, Luciferase