p2x2  (Alomone Labs)

Journal: The Journal of Neuroscience

Article Title: A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels

doi: 10.1523/JNEUROSCI.2390-15.2016

Figure Lengend 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.

Article Snippet: We have used previously described antibodies against the 5-HT3A ( ) and the 5-HT3B ( ) subunits to illustrate such a coexpression of native 5-HT3 and P2X2 receptors in neurons of the rat myenteric plexus using two independent couples of antibodies: rabbit anti-5-HT3A and guinea pig anti-P2X2 (Millipore) ( A ) and goat anti-5-HT3B and rabbit anti-P2X2 (Alomone Labs) ( B ).

Techniques: Immunohistochemistry

Journal: The Journal of Neuroscience

Article Title: A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels

doi: 10.1523/JNEUROSCI.2390-15.2016

Figure Lengend 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

Article Snippet: We have used previously described antibodies against the 5-HT3A ( ) and the 5-HT3B ( ) subunits to illustrate such a coexpression of native 5-HT3 and P2X2 receptors in neurons of the rat myenteric plexus using two independent couples of antibodies: rabbit anti-5-HT3A and guinea pig anti-P2X2 (Millipore) ( A ) and goat anti-5-HT3B and rabbit anti-P2X2 (Alomone Labs) ( B ).

Techniques: Transfection, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels

doi: 10.1523/JNEUROSCI.2390-15.2016

Figure Lengend 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.

Article Snippet: We have used previously described antibodies against the 5-HT3A ( ) and the 5-HT3B ( ) subunits to illustrate such a coexpression of native 5-HT3 and P2X2 receptors in neurons of the rat myenteric plexus using two independent couples of antibodies: rabbit anti-5-HT3A and guinea pig anti-P2X2 (Millipore) ( A ) and goat anti-5-HT3B and rabbit anti-P2X2 (Alomone Labs) ( B ).

Techniques: Transfection, Immunofluorescence, Fluorescence

Journal: The Journal of Neuroscience

Article Title: A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels

doi: 10.1523/JNEUROSCI.2390-15.2016

Figure Lengend 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

Article Snippet: We have used previously described antibodies against the 5-HT3A ( ) and the 5-HT3B ( ) subunits to illustrate such a coexpression of native 5-HT3 and P2X2 receptors in neurons of the rat myenteric plexus using two independent couples of antibodies: rabbit anti-5-HT3A and guinea pig anti-P2X2 (Millipore) ( A ) and goat anti-5-HT3B and rabbit anti-P2X2 (Alomone Labs) ( B ).

Techniques: Transfection, Immunofluorescence, Western Blot, Immunolabeling, Fluorescence

Journal: The Journal of Neuroscience

Article Title: A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels

doi: 10.1523/JNEUROSCI.2390-15.2016

Figure Lengend 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.

Article Snippet: We have used previously described antibodies against the 5-HT3A ( ) and the 5-HT3B ( ) subunits to illustrate such a coexpression of native 5-HT3 and P2X2 receptors in neurons of the rat myenteric plexus using two independent couples of antibodies: rabbit anti-5-HT3A and guinea pig anti-P2X2 (Millipore) ( A ) and goat anti-5-HT3B and rabbit anti-P2X2 (Alomone Labs) ( B ).

Techniques: Transfection, Immunofluorescence, Fluorescence, Expressing

Journal: The Journal of Neuroscience

Article Title: A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels

doi: 10.1523/JNEUROSCI.2390-15.2016

Figure Lengend 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

Article Snippet: We have used previously described antibodies against the 5-HT3A ( ) and the 5-HT3B ( ) subunits to illustrate such a coexpression of native 5-HT3 and P2X2 receptors in neurons of the rat myenteric plexus using two independent couples of antibodies: rabbit anti-5-HT3A and guinea pig anti-P2X2 (Millipore) ( A ) and goat anti-5-HT3B and rabbit anti-P2X2 (Alomone Labs) ( B ).

Techniques: Immunofluorescence, Fluorescence

Journal: The Journal of Neuroscience

Article Title: A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels

doi: 10.1523/JNEUROSCI.2390-15.2016

Figure Lengend 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

Article Snippet: We have used previously described antibodies against the 5-HT3A ( ) and the 5-HT3B ( ) subunits to illustrate such a coexpression of native 5-HT3 and P2X2 receptors in neurons of the rat myenteric plexus using two independent couples of antibodies: rabbit anti-5-HT3A and guinea pig anti-P2X2 (Millipore) ( A ) and goat anti-5-HT3B and rabbit anti-P2X2 (Alomone Labs) ( B ).

Techniques: Immunofluorescence, Transfection, Fluorescence, Cotransfection

Journal: The Journal of Physiology

Article Title: Intercellular Ca2+ signalling in the adult mouse cochlea

doi: 10.1113/JP276400

Figure Lengend Snippet: P2X2 receptor expression in the adult organ of Corti A , orthogonal projection of P2X2 receptor expression. P2X2 receptor immunofluorescence staining (green), myosin 7a (pink) and merged image. The hair cell cytoplasm is identified using myosin 7a as the label. B , surface view of P2X2 receptor immunohistochemistry in the IHC region of the adult mouse organ of Corti. C , P2X2 receptor staining in the OHC and Deiters’ cell region. Maximum projection image. Scale bar in all images = 10 μm.

Article Snippet: These were a P2X2 rabbit polyclonal antibody (APR‐003, Alomone Laboratories, Jerusalem, Israel) and a MYO7A monoclonal antibody, (MYO7A 138‐1, Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Immunohistochemistry

Journal: The Journal of Neuroscience

Article Title: Peripheral Inflammation Sensitizes P2X Receptor-Mediated Responses in Rat Dorsal Root Ganglion Neurons

doi: 10.1523/JNEUROSCI.22-01-00093.2002

Figure Lengend Snippet: CFA treatment enhances P2X2 and P2X3 receptor expression. A , Western blots for P2X2 and P2X3 receptors from ganglia of control rats ( CON ) and rats 5 d after CFA treatment. Actin control for each sample was given. B , Mean density relative to control rats

Article Snippet: Primary antibodies used were rabbit anti-P2X3 (1:3000; Neuromics Inc., Minneapolis, MN), rabbit anti-P2X1 and -P2X2 (1:200; Alomone Labs, Jerusalem, Israel), and mouse anti-actin (1:1000;Chemicon, Temecula, CA).

Techniques: Expressing, Western Blot

Journal: The Journal of Physiology

Article Title: Expression of P2X2 and P2X3 receptor subunits in rat carotid body afferent neurones: role in chemosensory signalling

doi: 10.1111/j.1469-7793.2001.00667.x

Figure Lengend Snippet: Expression of P2X2 and P2X3 subunits in tissue sections of petrosal ganglia and carotid body as revealed by confocal immunofluorescence A – C , represent the same tissue section from the petrosal ganglion of a 15-day-old rat after immunostaining with P2X2 and P2X3 antibodies. Localization of P2X2 ( A ) and P2X3 ( B ) subunits is revealed by Cy3 and FITC fluorescence, respectively; dual exposure in C shows overlap of P2X2 and P2X3 staining. D – F , the same section from the carotid body of a 14-day-old rat after similar immunostaining with P2X2 and P2X3 antibodies as in A – C . Note complete overlap of P2X2 and P2X3 immunostaining in nerve terminals, which are apposed to lobules of chemoreceptor cells. Calibration bar represents 40 μm in A – C , and 50 μm in D – F .

Article Snippet: The primary antibodies were: (i) anti-P2X2 (1:800 dilution), a rabbit polyclonal antibody raised against a highly purified peptide corresponding to amino acid residues 457-472 of rat P2X2 (Alomone Laboratories, Jerusalem, Israel); and (ii) anti-P2X3 (1:500 dilution), a guinea-pig polyclonal antiserum raised against amino acid residues 383-397 of rat P2X3 (Neuromics, Minneapolis, MN, USA).

Techniques: Expressing, Immunofluorescence, Immunostaining, Fluorescence, Staining

Journal: The Journal of Physiology

Article Title: Expression of P2X2 and P2X3 receptor subunits in rat carotid body afferent neurones: role in chemosensory signalling

doi: 10.1111/j.1469-7793.2001.00667.x

Figure Lengend Snippet: Detection of mRNA for P2X2, P2X3 and β-actin in isolated petrosal neurones RT-PCR was carried out on groups of isolated petrosal neurones using gene-specific primers for P2X2 and P2X3 receptors, and β-actin. Expected product sizes are (bp): P2X2, 357; P2X3, 326; β actin, 327. The marker lane (M) shows bands at 100 bp increments with the 600 bp fragment at increased intensity. In negative control reactions without RT (-) no PCR products were observed. PCR products were visualized with 2 % agarose gel stained with ethidium bromide and viewed under UV illumination.

Article Snippet: The primary antibodies were: (i) anti-P2X2 (1:800 dilution), a rabbit polyclonal antibody raised against a highly purified peptide corresponding to amino acid residues 457-472 of rat P2X2 (Alomone Laboratories, Jerusalem, Israel); and (ii) anti-P2X3 (1:500 dilution), a guinea-pig polyclonal antiserum raised against amino acid residues 383-397 of rat P2X3 (Neuromics, Minneapolis, MN, USA).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Negative Control, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

Journal: The Journal of Physiology

Article Title: Expression of P2X2 and P2X3 receptor subunits in rat carotid body afferent neurones: role in chemosensory signalling

doi: 10.1111/j.1469-7793.2001.00667.x

Figure Lengend Snippet: Whole-cell current-clamp recording and single-cell RT-PCR products A , phase-contrast micrograph of co-cultured type 1 or glomus cell (GC) cluster and juxtaposed petrosal neurone (PN); this configuration was used to test for functional neurones in electrophysiological experiments. Scale bar represents 10 μm. Ba , whole-cell current-clamp recording from a single petrosal neurone juxtaposed to a type 1 cell cluster in co-culture (similar to A ). For the period indicated by the horizontal bar, hypoxia ( P O 2 ∼ 5 mmHg) was applied by the switching of the extracellular perfusate for one equilibrated with 100 % N 2 . Following recording, the cell contents were aspirated and subjected to RT-PCR analysis as described in Methods. Bb , 2 % agarose gel showing RT-PCR products from the cell in Ba , typical of results obtained in seven cells examined. Expected product sizes are (bp): P2X2, 357; P2X3, 326; β-actin, 327. The marker lane (M) shows bands at 100 bp increments with the 600 bp fragment at increased intensity. Lane C shows a negative control where extracellular perfusate, aspirated from above the cells in the recording chamber, was subjected to RT-PCR analysis as described using primers for P2X2, P2X3 and β-actin.

Article Snippet: The primary antibodies were: (i) anti-P2X2 (1:800 dilution), a rabbit polyclonal antibody raised against a highly purified peptide corresponding to amino acid residues 457-472 of rat P2X2 (Alomone Laboratories, Jerusalem, Israel); and (ii) anti-P2X3 (1:500 dilution), a guinea-pig polyclonal antiserum raised against amino acid residues 383-397 of rat P2X3 (Neuromics, Minneapolis, MN, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Functional Assay, Co-Culture Assay, Agarose Gel Electrophoresis, Marker, Negative Control