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( 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 <t>ClustalX</t> algorithm and sequence analysis by PFAM database of protein families revealed the presence of WD40 domain exclusively in P2X6 subunit.
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( 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 <t>ClustalX</t> algorithm and sequence analysis by PFAM database of protein families revealed the presence of WD40 domain exclusively in P2X6 subunit.
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( 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.

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: ( 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: P2X receptors protein sequences were obtained from NCBI (P2X1, P51575; P2X2, Q9UBL9; P2X3, P56373; P2X4, Q99571; P2X5, Q93086; P2X6, O15547 and P2X7, Q99572), aligned using ClustalX algorithm in CLC Genomics Workbench v3.6.5 (CLC bio) and compared with full PFAM database plugin v22.0.

Techniques: Western Blot, Transfection, Control, Sequencing