Journal:

Article Title: Calcium signalling through nucleotide receptor P2X1 in rat portal vein myocytes

doi: 10.1111/j.1469-7793.2001.0339c.xd

Figure Lengend Snippet: A, amplified DNA fragments of P2X receptors (lanes 1–7) from rat brain (a) and rat portal vein myocytes (b) were separated on a 2 % agarose gel and visualized by staining with ethidium bromide. Lane 8, RNA from brain and portal vein myocytes in the absence of reverse transcriptase served as a negative control. Numbers on the left indicate molecular size standards in base pairs (bp). For RNA purification and PCR conditions, see Methods. B, immunostaining of P2X receptor subtypes in portal vein myocytes. Myocytes were stained with anti-P2X1 receptor (a) or anti-P2X4 receptor antibody (b) and vizualization was realized with a donkey anti-rabbit IgG FITC-conjugated antibody. In the absence of primary antibodies or after inactivation of the antibodies by their antigen peptides, only a faint background fluorescence was observed (not shown). Typical confocal sections were performed above the nucleus and therefore appeared spherical. Both P2X1 (a) and P2X4 (b) receptor subtypes were distributed throughout the confocal sections with a marked staining of P2X1 receptor subtype at the cell periphery.

Article Snippet: The rabbit anti-P2X1 and anti-P2X4 receptor antibodies (Alomone Labs, Jerusalem, Israel) were directed against polypeptides corresponding to residues 382–399 and 370–388 of the rat P2X1 and P2X4 receptors, respectively.

Techniques: Amplification, Agarose Gel Electrophoresis, Staining, Negative Control, Purification, Immunostaining, Fluorescence

Journal: Experimental neurology

Article Title: Neuroprotective and Neuro-rehabilitative Effects of Acute Purinergic Receptor P2X4 (P2X4R) Blockade after Ischemic Stroke

doi: 10.1016/j.expneurol.2020.113308

Figure Lengend Snippet: Plot indicates the effect of 5-BDBD at various concentrations (10 nM to 30 μM; log10 [nM] = −4.52 to −8) after P2X4R agonist ATP (1 μM) induced [Ca2+]i response. Calculated IC50 was 3.8 × 10−6 M.

Article Snippet: Primary antibodies used in western blot against P2X4R (Cat. 135341AP), BDNF (Cat. 256991AP), Beta-Actin (Cat. HRP-60008), and secondary antibody Anti-mouse (Cat. SA00002–1) were from Protein Tech, Rosemont, IL.

Techniques:

Journal: Experimental neurology

Article Title: Neuroprotective and Neuro-rehabilitative Effects of Acute Purinergic Receptor P2X4 (P2X4R) Blockade after Ischemic Stroke

doi: 10.1016/j.expneurol.2020.113308

Figure Lengend Snippet: Representative western blots are shown in the top panel. Effects of vehicle or 5-BDBD on pro and mature BDNF at (A) 3 days and (B) 30 days after stroke. C) Effects of vehicle and 5-BDBD treatment on pro/mature BDNF ratio 3 and 30 days after stroke. (D) P2X4R expression at 3 and 30 days after stroke were not different; Data are Mean ± SD (n=4–8 mice/group/time point). *p<0.05 for 5-BDBD vs vehicle by Student’s t-test. β-actin was used as internal control to normalize the data.

Article Snippet: Primary antibodies used in western blot against P2X4R (Cat. 135341AP), BDNF (Cat. 256991AP), Beta-Actin (Cat. HRP-60008), and secondary antibody Anti-mouse (Cat. SA00002–1) were from Protein Tech, Rosemont, IL.

Techniques: Western Blot, Expressing, Control

Journal: Experimental neurology

Article Title: Neuroprotective and Neuro-rehabilitative Effects of Acute Purinergic Receptor P2X4 (P2X4R) Blockade after Ischemic Stroke

doi: 10.1016/j.expneurol.2020.113308

Figure Lengend Snippet: Images (top) and quantification (bottom) of P2X4R expression (green) in lba-1 positive cells (red) from immunostaining of brain sections 3 days after stroke. 20x magnification. DAPI (blue) = 4′,6-diamidino-2-phenylindole (nuclear stain). Scale bar 20 μm. Data are Mean ± SD (n=3 mice/group). *p<0.05, 5-BDBD vs. Vehicle; Student’s t-test.

Article Snippet: Primary antibodies used in western blot against P2X4R (Cat. 135341AP), BDNF (Cat. 256991AP), Beta-Actin (Cat. HRP-60008), and secondary antibody Anti-mouse (Cat. SA00002–1) were from Protein Tech, Rosemont, IL.

Techniques: Expressing, Immunostaining, Staining

Journal: Experimental neurology

Article Title: Neuroprotective and Neuro-rehabilitative Effects of Acute Purinergic Receptor P2X4 (P2X4R) Blockade after Ischemic Stroke

doi: 10.1016/j.expneurol.2020.113308

Figure Lengend Snippet: Flow-sorted microglia and monocytes cells from Fig 5 were fixed (2% paraformaldehyde) and analyzed by an ImageStream cytometer. Using the IDEAS software package, all doublets or aggregated cells were excluded by gating on single cells. A) P2X4R and CD11b+ were focused and selected. Images of single cells (~300 cells/sample, N=3 per group) of the bright field and fluorescent channel (P2X4R-FITC and CD11b-APC-eFluor780) were acquired, and overlays were generated. Magnification=60x. B) Ratio of median fluorescence intensity (MFI) of P2X4R/CD11b was significantly reduced after 5-BDBD treatment in both microglia and monocytes. Further, a separate comparison of microglia vs. monocytes P2X4R MFI suggests reduced expression of P2X4R in microglia irrespective of treatment. Data are Mean ± SD; *p<0.05, 5-BDBD vs. Vehicle; #p<0.05, microglia vs. monocytes; Student’s t-test.

Article Snippet: Primary antibodies used in western blot against P2X4R (Cat. 135341AP), BDNF (Cat. 256991AP), Beta-Actin (Cat. HRP-60008), and secondary antibody Anti-mouse (Cat. SA00002–1) were from Protein Tech, Rosemont, IL.

Techniques: Cytometry, Software, Generated, Fluorescence, Comparison, Expressing

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: List of primary and secondary antibodies used in immunohistochemistry experiments.

Article Snippet: Confocal 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: Immunohistochemistry

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 < 0.05, **p < 0.01 (Student’s t -test for paired samples) represent significant differences when compared to the effects of ATP or CTP alone, respectively.

Article Snippet: Confocal 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 < 0.05 (Student’s t -test for paired samples) represent significant differences when compared to the effect of ATP alone.

Article Snippet: Confocal 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: Confocal 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: Confocal 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