rabbit anti p2x2 (Alomone Labs)
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Alomone Labs
rabbit anti p2x2
Rabbit Anti P2x2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti p2x2/product/Alomone Labs
Average 94 stars, based on 8 article reviews
Price from $9.99 to $1999.99
Rabbit Anti P2x2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti p2x2/product/Alomone Labs
Average 94 stars, based on 8 article reviews
Price from $9.99 to $1999.99
rabbit anti p2x2 - by Bioz Stars,
2022-08
94/100 stars
Images
1) Product Images from "A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels"
Article Title: A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels
Journal: The Journal of Neuroscience
doi: 10.1523/JNEUROSCI.2390-15.2016
Figure Legend Snippet: 5-HT 3 and P2X2 receptors are coexpressed in neurons of the myenteric plexus. Immunohistochemical detection of 5-HT 3A , 5-HT 3B , and P2X2 subunits in the rat intestinal tract at 4 d postnatal. A , 5-HT 3A (rabbit anti-5-HT 3A antibody; green) and P2X2 (guinea pig anti-P2X2 antibody; red). B , 5-HT 3B (goat anti 5-HT 3B antibody; green) and P2X2 (rabbit anti-P2X2 antibody; red). Both 5-HT 3 subunits colocalize with P2X2R in the same neurons (arrows). mp, Myenteric plexus; lm, longitudinal muscle; cm, circular muscle. Scale bar, 50 μm.
Techniques Used: Immunohistochemistry
Figure Legend Snippet: P2X2 receptor depletion inhibits 5-HT 3A receptor targeting toward distal neurites. Hippocampal neurons were transfected at 7 DIV with the 5-HT 3A R-HA alone or cotransfected with the 5-HT 3A R-HA plus siRNA against endogenous P2X2R or control siRNA for 48 h. A , Immunofluorescence detection of 5-HT 3A R-HA in representative neurons belonging to the P2X2-immunopositive subpopulation. Scale bar, 50 μm. B , 5-HT 3A R-HA immunofluorescence detection limits along the longest neurite (one neurite per neuron) were measured for each transfected neuron, and the neurons belonging to the P2X2-immunonegative and P2X2-immunopositive groups were represented separately. After RNA interference of endogenous P2X2R, the 5-HT 3A R-HA immunofluorescence detection limit was significantly lower in neurites of P2X2-immunopositive neurons only. Bars indicate mean ± SEM; n = 40–50. **** p
Techniques Used: Transfection, Immunofluorescence
Figure Legend Snippet: 5-HT 3A and P2X2 receptors colocalize in common surface clusters. A , 5-HT 3A R-HA was transfected alone or cotransfected with P2X2R-YFP in hippocampal neurons at 7 DIV. Immunofluorescence detection of both receptors (red represents 5-HT 3A R-HA; green represents P2X2-YFP) was performed after 48 h. The presence of transfected P2X2R in the same neurons enhanced the distal trafficking of 5-HT 3A R in all neurites, particularly in axons (arrows). Scale bar, 50 μm. B , Magnification of an axonal section reveals that 5-HT 3A R and P2X2R colocalize in common surface clusters (arrowheads). C , Fluorescence profiles of both labels along a polyline drawn through the surface clusters (boxed section). Scale bar, 5 μm.
Techniques Used: Transfection, Immunofluorescence, Fluorescence
Figure Legend Snippet: 5-HT 3A receptor distal targeting is dependent on endogenous P2X2 receptors. Hippocampal neurons were transfected at ( A ) 5 DIV, ( B ) 12 DIV, or ( C ) 7 DIV, with 5-HT 3A R-HA. Immunofluorescence detection was performed with the following: A , anti-HA (red) and anti-tubulin (green) antibodies; B , anti-HA antibodies without permeabilization (green), and then anti-HA antibodies after permeabilization (red); or C , anti-HA (red) and anti-P2X2 (green) antibodies. Scale bars, 50 μm. D , Endogenous P2X2R (arbitrary units) were quantified by Western blot (black line) in hippocampal cultures at the indicated times (DIV). Surviving neurons were counted for each condition (red line), and the amount of endogenous P2X2R was divided by the number of live neurons in the culture at each time (blue line, endogenous P2X2/surviving neurons). E , The intensity of mean endogenous anti-P2X2R immunofluorescence (arbitrary units) measured on neuron somas was plotted versus the distance of the 5-HT 3A -HA detection limit (above background) along the longest immunolabeled neurite at 7 DIV ( C ). The cumulated frequency distribution of P2X2R mean fluorescence intensity on somas (inset) revealed the existence of two populations of neurons ( n = 51, bimodal distribution, median = 50): P2X2 immunonegative (68.3%, μ 1 = 47, quartiles = 40) and P2X2 immunopositive (31.6%, μ 2 = 121, quartiles = 110). Only P2X2R-immunopositive neurons (example in C ) expressed 5-HT 3A R distally (linear regression, r 2 = 0.71, p
Techniques Used: Transfection, Immunofluorescence, Western Blot, Immunolabeling, Fluorescence
Figure Legend Snippet: The majority of hippocampal neurons in culture do not address 5-HT 3A receptors distally. Hippocampal neurons were transfected at 7 DIV with 5-HT 3A -HA, untagged 5-HT 3A , 5-HT 1A -eGFP, sst2A-eGFP, or P2X2-YFP subunits. A , Immunofluorescence was performed with anti-HA, anti-5-HT 3A , or anti-GFP antibodies (to enhance GFP and YFP signals). B , Cumulated fluorescence intensities along the longest neurite were plotted for a representative sample of 10 neurons from each transfected culture. Whereas 70% of 5-HT 3A R expressing neurons did not address the receptor distally in neurites, nearly 100% of transfected neurons addressed 5-HT 1A R in the entire dendritic tree and nearly 100% of neurons addressed sst2AR and P2X2R in the entire dendritic and axonal arborizations. Scale bar, 50 μm.
Techniques Used: Transfection, Immunofluorescence, Fluorescence, Expressing
Figure Legend Snippet: Specificity of the 5-HT 3A and P2X2 receptor interaction. Hippocampal neurons were cotransfected at 7 DIV with 5-HT 3A -HA and P2X2-YFP, 5-HT 1A -eGFP, sst2A-eGFP, myc-GABAc (ρ1), myc-MT2, myc-GluA1, myc-GluA2, or myc-NR2A subunits. A , Immunofluorescence detection was performed after 48 h (red represents anti-HA; green represents anti-eGFP or anti-myc). Scale bars, 20 μm. ROI were chosen within the dendritic trees to select cluster-rich areas and avoid the center of large neurites, and colocalization was monitored with the JACoP plugin of ImageJ (manual thresholding). Corresponding fluorograms for each cotransfected couple of receptors are represented on the right panels. B , Bars represent mean ± SEM values of Mander's M1 coefficients (percentage of 5-HT 3A R-HA fluorescence overlapping with cotransfected receptor's fluorescence). Significant differences appeared only between interacting (P2X2) and noninteracting subunits (one-way ANOVA with Dunnett's Multiple Comparison post hoc test); n = 4–8. **** p
Techniques Used: Immunofluorescence, Fluorescence
Figure Legend Snippet: P2X2 receptors induce 5-HT 3A receptor targeting in axons. Hippocampal neurons were cotransfected at 4–5 DIV with 5-HT 3A R-HA plus eGFP or 5-HT 3A R-HA plus P2X2-YFP. A , Immunofluorescence detection (red represents anti-HA; green represents anti-eGFP) was performed 48 h after transfection. Arrows indicate the axons. Scale bar, 50 μm. B , Cumulated fluorescence intensity profiles of 5-HT 3A R-HA along the axons (longest path, n = 30) for 5-HT 3A R-HA plus eGFP (blue) or 5-HT 3A R-HA plus P2X2-YFP (red). C , Cumulated fluorescence intensity profiles of eGFP and P2X2-YFP along the same axons ( n = 30) for 5-HT 3A R-HA plus eGFP (green) or 5-HT 3A R-HA plus P2X2-YFP (orange). D , Quantification of the number of neurons exhibiting 5-HT 3A R-HA immunofluorescence at distances above (distal) or below (proximal) 100 μm from somas for 5-HT 3A R-HA plus eGFP or 5-HT 3A R-HA plus P2X2-YFP. Bars represent the relative proportions of the two groups (mean ± SEM) for each cotransfection condition (two-way ANOVA, n = 324 and n = 399, respectively, p
Techniques Used: Immunofluorescence, Transfection, Fluorescence, Cotransfection
2) Product Images from "Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance"
Article Title: Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance
Journal: Development (Cambridge, England)
doi: 10.1242/dev.150342
Figure Legend Snippet: Gustatory ganglion cells that innervate taste buds express Shh-tdTomato. (A) Shh CreERT2 ; R26R tdTomato (Shh-tdTomato) mice were given tamoxifen daily for 4 days and harvested at 10, 60 or 85 days. (B-D) In Shh-tdTomato + tongues, punctate signal (red) is evident in FFP at all times (arrowheads); at 60 and 85 days (C,D) gustatory innervation (arrows) associated with FFP (arrowheads) is also labeled. (E) Gustatory neurons in the geniculate ganglion (gVII) innervate FFP via the chorda tympani (CT) nerve, and project to the nucleus of the solitary tract (NST) in the brainstem. The greater superficial petrosal (GSP) nerve innervates soft palate taste buds (not shown). gVII neurons project centrally to the NST. (F-H) Cells in gVII express tdTomato at all time points. (I-K‴) At 60 days, PGP9.5 + nerve fibers (white in J,K″; green in K,K‴) innervate taste buds and adjacent FF epithelium, whereas Shh-tdTomato + neurites (white in I,K′; red in K,K‴), which are also PGP9.5 + , innervate taste buds exclusively (K′-K‴, cyan arrowheads). (L-N‴) P2X2 + taste fibers (white in M,N′; green in N,N‴) express Shh-tdTomato (white in L,N′; red in N,N‴; cyan arrowheads). Boxes in K and N are shown at higher magnification in K′-K‴ and N′-N‴, respectively. Sparse bright lineage-labeled taste cells are detected at later times (I,L, arrows), and are distinguishable from dimmer Shh-tdTomato + neurites (I,L, arrowheads within taste buds). Nuclei are counterstained with Draq5 (blue). B-D and F-H are images of whole tongue and ganglia. I-N‴ are compressed confocal z -stacks. Scale bars: 1 mm in B-D; 150 μm in F-H; 10 μm in I-N‴.
Techniques Used: Mouse Assay, Labeling
Figure Legend Snippet: Genetic deletion of Shh in Shh + cells, including gustatory neurons, minimally affects taste buds. (A) Shh CreERT2/flox ; R26R tdTomato (Shh-ShhcKO) and genetic control mice were given tamoxifen for 4 days and harvested at 35 days. (B) In tamoxifen-treated Shh-ShhcKO mice, Shh is deleted permanently from ganglion cells and nerves (indicated by the cross), but transiently from taste buds (see text). (C,D) tdTomato reports Shh deletion in tongue (C) and gVII (D) of mutant mice. (E) Quantitative PCR reveals that neither Shh nor Gli1 expression is significantly reduced in mutant tongue epithelium. (F) Expression of Shh , but not Gli1, is significantly reduced in mutant gVII. (G,H) Typical FFP number does not differ between mutants and controls (G), although atypical FFP increase in mutants (H). (I,J) The size of taste buds in typical (I) and atypical (J) FFP in mutants does not differ from controls. (K) Shh-tdTomato + neurites (red) innervate a taste bud (K8 + , green) in an atypical FFP in a Shh-ShhcKO mouse (Shh-descendent taste cell, arrow). (L) The proportion of FFP innervated by P2X2 + fibers is not affected by Shh-ShhcKO. (M,N) P2X2 + innervation density of taste buds in typical (M) and atypical (N) FFP does not differ between controls and Shh-ShhcKO mice. Nuclei counterstained with Draq5 (blue); white dashed lines delimit basement membrane; white solid lines delimit epithelial surface. C and D are images of whole tongue and gVII. K is a compressed confocal z -stack. Scale bars: 1 mm in C; 150 μm in D; 10 μm in K. n =3-5 mice per condition. Data are mean±s.d., except E and F, which are mean±s.e.m; I and N represent the median with interquartile range. Results were analyzed using Student's t -test (E-H,J,L,M) or Mann–Whitney U-test (I,N). *** P
Techniques Used: Mouse Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY
3) Product Images from "Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance"
Article Title: Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance
Journal: Development (Cambridge, England)
doi: 10.1242/dev.150342
Figure Legend Snippet: Gustatory ganglion cells that innervate taste buds express Shh-tdTomato. (A) Shh CreERT2 ; R26R tdTomato (Shh-tdTomato) mice were given tamoxifen daily for 4 days and harvested at 10, 60 or 85 days. (B-D) In Shh-tdTomato + tongues, punctate signal (red) is evident in FFP at all times (arrowheads); at 60 and 85 days (C,D) gustatory innervation (arrows) associated with FFP (arrowheads) is also labeled. (E) Gustatory neurons in the geniculate ganglion (gVII) innervate FFP via the chorda tympani (CT) nerve, and project to the nucleus of the solitary tract (NST) in the brainstem. The greater superficial petrosal (GSP) nerve innervates soft palate taste buds (not shown). gVII neurons project centrally to the NST. (F-H) Cells in gVII express tdTomato at all time points. (I-K‴) At 60 days, PGP9.5 + nerve fibers (white in J,K″; green in K,K‴) innervate taste buds and adjacent FF epithelium, whereas Shh-tdTomato + neurites (white in I,K′; red in K,K‴), which are also PGP9.5 + , innervate taste buds exclusively (K′-K‴, cyan arrowheads). (L-N‴) P2X2 + taste fibers (white in M,N′; green in N,N‴) express Shh-tdTomato (white in L,N′; red in N,N‴; cyan arrowheads). Boxes in K and N are shown at higher magnification in K′-K‴ and N′-N‴, respectively. Sparse bright lineage-labeled taste cells are detected at later times (I,L, arrows), and are distinguishable from dimmer Shh-tdTomato + neurites (I,L, arrowheads within taste buds). Nuclei are counterstained with Draq5 (blue). B-D and F-H are images of whole tongue and ganglia. I-N‴ are compressed confocal z -stacks. Scale bars: 1 mm in B-D; 150 μm in F-H; 10 μm in I-N‴.
Techniques Used: Mouse Assay, Labeling
Figure Legend Snippet: Genetic deletion of Shh in Shh + cells, including gustatory neurons, minimally affects taste buds. (A) Shh CreERT2/flox ; R26R tdTomato (Shh-ShhcKO) and genetic control mice were given tamoxifen for 4 days and harvested at 35 days. (B) In tamoxifen-treated Shh-ShhcKO mice, Shh is deleted permanently from ganglion cells and nerves (indicated by the cross), but transiently from taste buds (see text). (C,D) tdTomato reports Shh deletion in tongue (C) and gVII (D) of mutant mice. (E) Quantitative PCR reveals that neither Shh nor Gli1 expression is significantly reduced in mutant tongue epithelium. (F) Expression of Shh , but not Gli1, is significantly reduced in mutant gVII. (G,H) Typical FFP number does not differ between mutants and controls (G), although atypical FFP increase in mutants (H). (I,J) The size of taste buds in typical (I) and atypical (J) FFP in mutants does not differ from controls. (K) Shh-tdTomato + neurites (red) innervate a taste bud (K8 + , green) in an atypical FFP in a Shh-ShhcKO mouse (Shh-descendent taste cell, arrow). (L) The proportion of FFP innervated by P2X2 + fibers is not affected by Shh-ShhcKO. (M,N) P2X2 + innervation density of taste buds in typical (M) and atypical (N) FFP does not differ between controls and Shh-ShhcKO mice. Nuclei counterstained with Draq5 (blue); white dashed lines delimit basement membrane; white solid lines delimit epithelial surface. C and D are images of whole tongue and gVII. K is a compressed confocal z -stack. Scale bars: 1 mm in C; 150 μm in D; 10 μm in K. n =3-5 mice per condition. Data are mean±s.d., except E and F, which are mean±s.e.m; I and N represent the median with interquartile range. Results were analyzed using Student's t -test (E-H,J,L,M) or Mann–Whitney U-test (I,N). *** P
Techniques Used: Mouse Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY
4) Product Images from "Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium"
Article Title: Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium
Journal: Development (Cambridge, England)
doi: 10.1242/dev.107631
Figure Legend Snippet: Fungiform taste buds are innervated by P2X2 + and PGP9.5 + nerve fibers, whereas ectopic taste buds form in lingual epithelium innervated only by PGP9.5 + fibers. (A) A fungiform taste bud is innervated by P2X2 + taste fibers, whereas ectopic taste buds (B)
Techniques Used:
5) Product Images from "Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium"
Article Title: Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium
Journal: Development (Cambridge, England)
doi: 10.1242/dev.107631
Figure Legend Snippet: Fungiform taste buds are innervated by P2X2 + and PGP9.5 + nerve fibers, whereas ectopic taste buds form in lingual epithelium innervated only by PGP9.5 + fibers. (A) A fungiform taste bud is innervated by P2X2 + taste fibers, whereas ectopic taste buds (B)
Techniques Used:
6) Product Images from "Developmentally dynamic colocalization patterns of DSCAM with adhesion and synaptic proteins in the mouse retina"
Article Title: Developmentally dynamic colocalization patterns of DSCAM with adhesion and synaptic proteins in the mouse retina
Journal: Molecular Vision
doi:
Figure Legend Snippet: Limited colocalization of DSCAM and synaptic markers detected in the adult retina. Analysis of colocalization between DSCAM and C-Terminal Binding Protein-2 (CTBP2; A ), NMDA R1 (NR1) ( B ), Synapsin ( C ), Neuroligin ( D ) NMDA 2B ( E ), NMDA 2AB ( F ) Synapse Associated Protein 102 (SAP102; G ), Shank ( H ), P2X2 ( I ), P2X7 ( J ), Glutamate Receptor 4 (GluR4; K ), GABAaδ2 ( L ), Calcium/Calmodulin-Dependent Serine Protein Kinase (CASK; M ), Bassoon ( N ), Vesiclar Glutamate Transporter 3 (vglut3; O ), Syntaxin 2 ( P ), RIM 2 ( Q ), Glycine Receptor ( R ), Glutamate Receptor 1 (GluR1; S ), Oxidative Phosphorylation marker ( T ), Membrane Associated Guanylate Kinase 2 (MAGI2; U ), Connexin 36 ( V ), 14-3-3 ε ( W ), Syntaxin 4 ( X ), ZO-1-Associated Nucleic Acid-Binding Protein (ZONAB; Y ), Membrane Associated Guanylate Kinase 3 (MAGI3; Z ), Zonula Occludens-2 ( AA ) or 14-3-3 ξ ( BB ). CC and DD , Dispersed cultures of retinal ganglion cells were stained with antibodies to DSCAM, PSD95 and GABAa∂2. Only rare examples of DSCAM overlap with synaptic markers were observed ( CC arrow). The scale bar in DD is equivalent to 53.25 µm in A - CC and 7.9 µm in insets from A - CC . The scale bar in DD is equivalent to 15.8 µm in CC and 35.5 µm in DD .
Techniques Used: Binding Assay, Marker, Staining
7) Product Images from "P2X2 and P2X5 Subunits Define a New Heteromeric Receptor with P2X7-Like Properties"
Article Title: P2X2 and P2X5 Subunits Define a New Heteromeric Receptor with P2X7-Like Properties
Journal: The Journal of Neuroscience
doi: 10.1523/JNEUROSCI.6332-11.2012
Figure Legend Snippet: P2X5 subunits interact with P2X2 subunits at the plasma membrane. A , Rescue of cell surface expression of the P2X2–K366A trafficking deficient mutant by other subunits. P2X2–K366A carrying a Flag tag in the extracellular loop was expressed alone or in combination with each of the seven wild-type P2X subunits. Membrane expression was measured using a chemiluminescent assay. Cell surface expression of P2X2–K366A is rescued upon coexpression with P2X2, P2X3, P2X4, and P2X5 subunits. B , Membrane detection of the P2X5–K372A mutant carrying an extracellular Flag tag is increased when coexpressed with P2X1, P2X2, and P2X4 and P2X5 subunits. Note that because the surface expression of P2X5–K372 mutant is very low compared withP2X2–K366A, the changes appear much higher. Results are shown as mean ± SEM of at least three independent experiments. ** p
Techniques Used: Expressing, Mutagenesis, FLAG-tag
Figure Legend Snippet: Stoichiometry of P2X2 and P2X5 interactions as assessed by BiFC and BRET/BiFC. A , Bimolecular fluorescence complementation between P2X subunits. P2X subunits fused to either the amino or carboxyl half of YFP (YN or YC) were transfected alone or in combination in HEK cells. Recomplemented fluorescence was observed by microscopy. BiFC was observed between homomeric P2X2, P2X4, and P2X5 subunits, as well as between heteromeric P2X2 and P2X5 subunits, but not between P2X2 and P2X4 subunits. Scale bar, 10 μm. B , Combination of BRET and BiFC reveals the stoichiometry of the heteromeric P2X2/P2X5 assembly. Top, Diagram illustrating the approach. BRET is only observed between one P2X2-Luc subunit and any other combinations of two P2X-hemi-YFP subunits. BRET titration curves between P2X2-Luc (left) or P2X5-Luc (right) cotransfected with P2X2, P2X5, RAMP1, and CRL fused to hemi-YFP. Specific BRET signals were observed between P2X2 and P2X5 subunits for each experimental condition, demonstrating the existence of heteromeric receptors with two different stoichiometries. Data are expressed as mean ± SEM of at least N = 3 experiments.
Techniques Used: Bimolecular Fluorescence Complementation Assay, Bioluminescence Resonance Energy Transfer, Fluorescence, Transfection, Microscopy, Titration
Figure Legend Snippet: P2X5 and P2X2 subunits interact in neuronal tissues. A , Physical interactions of P2X2 and P2X5 subunits in brain tissues. P2X2 or P2X5 subunits were immunoprecipitated from mouse brain membrane and different subunits detected with specific antibodies. Left panel represents immunoprecipitation from total brain membrane proteins, right panel from brainstem. Note that P2X5 is not detectable from the total protein extract. Blots are representative of three independent experiments. Asterisks indicate the antibody heavy chains. B , Coimmunolocalization of P2X2 and P2X5 subunits in peripheral and central neurons. Representative images of immunohistochemistry performed on slices from DRG, spinal cord, or mid pons. P2X2 and P2X5 are stained in red and green, respectively. Note that in each structure only a subset of neurons coexpresses both subunits. Scale bar, 40 μm.
Techniques Used: Immunoprecipitation, Immunohistochemistry, Staining
8) Product Images from "P2X2 and P2X5 Subunits Define a New Heteromeric Receptor with P2X7-Like Properties"
Article Title: P2X2 and P2X5 Subunits Define a New Heteromeric Receptor with P2X7-Like Properties
Journal: The Journal of Neuroscience
doi: 10.1523/JNEUROSCI.6332-11.2012
Figure Legend Snippet: P2X5 subunits interact with P2X2 subunits at the plasma membrane. A , Rescue of cell surface expression of the P2X2–K366A trafficking deficient mutant by other subunits. P2X2–K366A carrying a Flag tag in the extracellular loop was expressed alone or in combination with each of the seven wild-type P2X subunits. Membrane expression was measured using a chemiluminescent assay. Cell surface expression of P2X2–K366A is rescued upon coexpression with P2X2, P2X3, P2X4, and P2X5 subunits. B , Membrane detection of the P2X5–K372A mutant carrying an extracellular Flag tag is increased when coexpressed with P2X1, P2X2, and P2X4 and P2X5 subunits. Note that because the surface expression of P2X5–K372 mutant is very low compared withP2X2–K366A, the changes appear much higher. Results are shown as mean ± SEM of at least three independent experiments. ** p
Techniques Used: Expressing, Mutagenesis, FLAG-tag
Figure Legend Snippet: Stoichiometry of P2X2 and P2X5 interactions as assessed by BiFC and BRET/BiFC. A , Bimolecular fluorescence complementation between P2X subunits. P2X subunits fused to either the amino or carboxyl half of YFP (YN or YC) were transfected alone or in combination in HEK cells. Recomplemented fluorescence was observed by microscopy. BiFC was observed between homomeric P2X2, P2X4, and P2X5 subunits, as well as between heteromeric P2X2 and P2X5 subunits, but not between P2X2 and P2X4 subunits. Scale bar, 10 μm. B , Combination of BRET and BiFC reveals the stoichiometry of the heteromeric P2X2/P2X5 assembly. Top, Diagram illustrating the approach. BRET is only observed between one P2X2-Luc subunit and any other combinations of two P2X-hemi-YFP subunits. BRET titration curves between P2X2-Luc (left) or P2X5-Luc (right) cotransfected with P2X2, P2X5, RAMP1, and CRL fused to hemi-YFP. Specific BRET signals were observed between P2X2 and P2X5 subunits for each experimental condition, demonstrating the existence of heteromeric receptors with two different stoichiometries. Data are expressed as mean ± SEM of at least N = 3 experiments.
Techniques Used: Bimolecular Fluorescence Complementation Assay, Bioluminescence Resonance Energy Transfer, Fluorescence, Transfection, Microscopy, Titration
Figure Legend Snippet: P2X5 and P2X2 subunits interact in neuronal tissues. A , Physical interactions of P2X2 and P2X5 subunits in brain tissues. P2X2 or P2X5 subunits were immunoprecipitated from mouse brain membrane and different subunits detected with specific antibodies. Left panel represents immunoprecipitation from total brain membrane proteins, right panel from brainstem. Note that P2X5 is not detectable from the total protein extract. Blots are representative of three independent experiments. Asterisks indicate the antibody heavy chains. B , Coimmunolocalization of P2X2 and P2X5 subunits in peripheral and central neurons. Representative images of immunohistochemistry performed on slices from DRG, spinal cord, or mid pons. P2X2 and P2X5 are stained in red and green, respectively. Note that in each structure only a subset of neurons coexpresses both subunits. Scale bar, 40 μm.
Techniques Used: Immunoprecipitation, Immunohistochemistry, Staining
