p2x4 antibody  (Alomone Labs)

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

doi: 10.3390/ijms222010970

Figure Lengend Snippet: Proposed mechanism for EV action on microglial cells. EVs carrying MFG-E8 proteins associated with phosphatidylserine exposed on the outer membrane are recognized by the αVβ3/αVβ5 integrin receptors of microglial cells and trigger lipid raft formation, interaction with P2X4 receptors, and possibly other molecules enriched in the lipid rafts such as components of the TLR4 multireceptor complex. These events lead to the upregulation of intracellular Ca 2+ , release of ATP, and increased motility of microglia.

Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

doi: 10.3390/ijms222010970

Figure Lengend Snippet: Co-immunoprecipitation of MFG-E8 and P2X4. Representative Western blots showing co-immunoprecipitation of MFG-E8 and P2X4R proteins in human microglial cells treated (or not) with EVs for 2 h, + indicates treatment with appropriate antibody. Full blots are available in Supplementary Figure S4 .

Article Snippet: Cells were then incubated with a pair of primary antibodies against MFG-E8 (Santa Cruz Biotechnology; sc-217574) and P2X4 (Alomone Labs; APR-002) (dilution 1:500) for 1 h at RT, washed two times for 5 min with wash buffer A, and incubated with a mix of PLA anti-rabbit PLUS and PLA anti-mouse MINUS oligonucleotide-conjugated secondary antibodies for 1 h at 37 °C.

Techniques: Immunoprecipitation, Western Blot

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.

Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

Techniques: Expressing, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .

Article Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-024, extracellular loop, Alomone) and of the HCN4 channel (Agp-004, Alomone) in the rat sinoatrial node (SAN); images obtained in right atria (RA) and right ventricle (RV) are also shown for comparison.

Techniques: Activity Assay, Inhibition

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: The negative chronotropic effect of ATP is mediated by activation of P2X4. The negative chronotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X7 receptor antagonist, A438079 (3 µM, A ), the P2X4 receptor antagonist, 5-BDBD (10 µM, B ) and the positive allosteric modulator of the P2X4 receptor, ivermectin (30 µM, C ). The negative chronotropic effect of CTP (1 mM, D ) either in the absence or in the presence of 5-BDBD (10 µM), is also shown for comparison. Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from baseline; horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

Article Snippet: Localization of P2X4, NCX1, and HCN4 Proteins in the Rat HeartConfocal micrographs shown in demonstrate that P2X4 receptor protein is expressed in the plasma membrane of cardiomyocytes of all assayed regions of the rat heart; in these experiments we used a knock-out validated antibody targeting the amino acid residues 370–388 of the C-terminus of the rat P2X4 receptor (Apr-002 from Alomone).

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: Selective blockage of P2X4 and of NCX transporter partially offsets the negative inotropic effect of ATP in paced rat ventricular strips. The negative inotropic effect of ATP (100 µM) was tested either in the absence or in the presence of the P2X4 receptor antagonist, 5-BDBD (10 µM, A and B ) and of the NCX inhibitor, KB-R7943 (3 µM, C and D ). Represented are box-and-whiskers plots, with whiskers ranging from minimum to maximum values calculated as a percentage (%) of variation from the baseline isometric tension of RV strips, measured as the active tension (mN/mg of wet tissue weight, panels A and C ) and the derivative of developed force over time (+dF/dt, mN/s, panels B and D ); horizontal lines inside boxes indicate the corresponding medians. Each data point represents the result of a single experiment; data from the same experiment are connected by lines. *p

Article Snippet: Localization of P2X4, NCX1, and HCN4 Proteins in the Rat HeartConfocal micrographs shown in demonstrate that P2X4 receptor protein is expressed in the plasma membrane of cardiomyocytes of all assayed regions of the rat heart; in these experiments we used a knock-out validated antibody targeting the amino acid residues 370–388 of the C-terminus of the rat P2X4 receptor (Apr-002 from Alomone).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: Representative confocal micrographs showing the immunolocalization of the P2X4 receptor (Apr-002, C-terminus, Alomone) and NCX1 (Anx-011, Alomone) protein in the sinoatrial node (SAN), right atria (RA) and right ventricle (RV). The SAN was identified based on its low Cx43 (green) and high HCN4 (magenta) protein expression (left hand-side images). Images were taken from whole-mount heart preparations including the three analyzed regions, SAN, RA and RV. Dashed lines represent boundaries of the SAN. The pulmonary parenchyma was used as a structural support to facilitate immunostaining of myocardial sections and it is visible in the bottom right quadrant of each SAN image. White arrows indicate blood vessels including the SAN artery. Scale bar 30 µm. Images are representative of three different individuals.

Article Snippet: Localization of P2X4, NCX1, and HCN4 Proteins in the Rat HeartConfocal micrographs shown in demonstrate that P2X4 receptor protein is expressed in the plasma membrane of cardiomyocytes of all assayed regions of the rat heart; in these experiments we used a knock-out validated antibody targeting the amino acid residues 370–388 of the C-terminus of the rat P2X4 receptor (Apr-002 from Alomone).

Techniques: Expressing, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy

doi: 10.3389/fphar.2019.01103

Figure Lengend Snippet: The mechanism underlying the dual P2X4 receptor-mediated effects on cardiac chronotropy and inotropy implicates downstream modulation of NCX activity (digitalis-like phenomenon). Besides ion fluxes carried by pacemaker HCN channels (not represented), the unstable resting membrane potential and the spontaneous firing of SAN cardiomyocytes are attributed mainly to the electrogenic NCX transport operating in the forward Ca 2+ -extrusion mode. Na + influx through the P2X4 receptor pore dissipates the electrochemical gradient of this ion across the plasma membrane leading to inhibition and/or reversion of the NCX pacemaker current. This may justify slowing down of SAN cells depolarizations and the negative chronotropic effect of ATP. Likewise, intracellular Ca 2+ accumulation due both (1) to Ca 2+ influx through the P2X4 receptor pore, and (2) to reversal of NCX activity may explain the positive inotropic effect of the P2X4 receptor in paced ventricular cardiomyocytes. Figure composition used elements from Servier Medical Art .

Article Snippet: Localization of P2X4, NCX1, and HCN4 Proteins in the Rat HeartConfocal micrographs shown in demonstrate that P2X4 receptor protein is expressed in the plasma membrane of cardiomyocytes of all assayed regions of the rat heart; in these experiments we used a knock-out validated antibody targeting the amino acid residues 370–388 of the C-terminus of the rat P2X4 receptor (Apr-002 from Alomone).

Techniques: Activity Assay, Inhibition

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: Brain sections were pretreated for 1 h with 1% bovine serum albumin (BSA) (Sigma-Aldrich), 5% FBS, and 0.2% Triton X-100 (Sigma-Aldrich) in PBS, and subsequently incubated with rabbit polyclonal antibodies against P2X6 (Alomone Labs, 1/50), P2X4 (Alomone Labs, 1/500), and/or P2X2 (Alomone Labs, 1/200) receptors.

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: 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: Brain sections were pretreated for 1 h with 1% bovine serum albumin (BSA) (Sigma-Aldrich), 5% FBS, and 0.2% Triton X-100 (Sigma-Aldrich) in PBS, and subsequently incubated with rabbit polyclonal antibodies against P2X6 (Alomone Labs, 1/50), P2X4 (Alomone Labs, 1/500), and/or P2X2 (Alomone Labs, 1/200) receptors.

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