Journal: The Journal of Biological Chemistry

Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

doi: 10.1074/jbc.M111.253153

Figure Lengend Snippet: Isolation of P2X1 receptors and associated proteins. A , anti-FLAG antibody-agarose beads were incubated with lysates of either native HEK293 cells or HEK293 cells stably expressing human P2X1 His-FLAG-tagged receptor. The agarose bead-protein complex was loaded onto a column and flow-through ( FT ) collected. Beads were washed four times and a sample of eluate kept (washes 1–4). 3× FLAG peptide (0.1 mg/ml) eluted 10 1-ml fractions (elutions 1–10) followed by two fractions eluted with 0.1 m glycine (pH 3.5) (elutions 11 and 12). Fractions were analyzed by Western blotting and probed with P2X1 antibody. No immunoreactivity was observed in HEK native cell lysate fractions, but strong immunoreactivity was observed in fractions E2–E8 from HEK293 cells expressing P2X1 receptor. B , fractions E2–E8 were pooled, concentrated, and run on SDS-PAGE after which the gel was stained for the presence of proteins. Cytoskeletal proteins are listed on the right as identified by mass spectrometery from HEK293 cells expressing P2X1 receptor lane gel slices. No proteins were identified from the HEK native receptor lane gel slices.

Article Snippet: Membranes were then processed with the primary anti-P2X1 receptor antibodies (1:1000; Alomone).

Techniques: Isolation, Incubation, Stable Transfection, Expressing, Western Blot, SDS Page, Staining

Journal: The Journal of Biological Chemistry

Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

doi: 10.1074/jbc.M111.253153

Figure Lengend Snippet: Inhibition of P2X1 receptor-mediated currents by actin cytoskeleton disruption. Effects of actin cytoskeleton disruption by cytochalasin D ( Cyto ) or latrunculin A ( Lat ) on P2X1 receptor currents were tested for recombinant human P2X1 receptors expressed in HEK293 cells ( A , C , E , and F ) and native rat smooth muscle P2X1 receptors ( D ). Application of cytochalasin D (5 μ m , gray symbols ) in external solution reduced the amplitude of α,β-meATP-evoked currents (10 μ m , applied every 5 min) in the permeabilized patch recording configuration ( A and B ). 20-min treatment with cytochalasin D before stimulating the P2X1 receptor is shown as a star . In the whole cell recording configuration P2X1 receptor currents in HEK293 cells or rat smooth muscle cells treated with cytochalasin D (500 n m ,1 h) or latrunculin A (500 n m , 1 h) were reduced ( C–E ). Surface biotinylation showed that actin cytoskeleton disruption by cytochalasin D (500 n m ,1 h) did not change either the total or surface level of P2X1 receptors expression in HEK293 cells ( F ). ***, p < 0.001.

Article Snippet: Membranes were then processed with the primary anti-P2X1 receptor antibodies (1:1000; Alomone).

Techniques: Inhibition, Recombinant, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

doi: 10.1074/jbc.M111.253153

Figure Lengend Snippet: Stabilization of the actin cytoskeleton by jasplakinolide does not prevent the rundown of P2X1 receptor-mediated currents in the whole cell recording configuration. In the permeabilized patch recording configuration, which supports intracellular signaling pathways, reproducible α,β-meATP (10 μ m )-evoked currents were observed every 5 min both for control cells and cells treated with jasplakinolide (30 n m , 1 h) to stabilize the actin cytoskeleton. In the whole cell recording configuration P2X1 receptor currents decreased in amplitude following repeated application of agonist at 5-min intervals. This rundown was unaffected by jasplakinolide treatment.

Article Snippet: Membranes were then processed with the primary anti-P2X1 receptor antibodies (1:1000; Alomone).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

doi: 10.1074/jbc.M111.253153

Figure Lengend Snippet: Alterations in actin cytoskeleton status lead to changes in the fraction of immobile P2X1 receptors. A , representative snapshots and traces of FRAP for HEK293 cells expressing P2X1-EGFP receptors in control conditions (area bleached is shown by the circle , and numbers refer to the time of image, and star indicates the bleach event). The time course of FRAP is shown under control conditions and following treatment with either cytochalasin D ( Cyto , 500 n m , 1 h) or jasplakinolide ( Jasp , 30 n m , 1 h). B , summary data of the immobile fraction for P2X1 receptors after stabilization of actin cytoskeleton by jasplakinolide and after disruption of actin cytoskeleton by cytochalasin D ( n = 9–20). *, p < 0.05.

Article Snippet: Membranes were then processed with the primary anti-P2X1 receptor antibodies (1:1000; Alomone).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

doi: 10.1074/jbc.M111.253153

Figure Lengend Snippet: Identification of the regions of the P2X1 receptor that contribute to actin sensitivity. A , schematic representation of chimeric P2X1/P2X2 receptors. P2X1/2aα and P2X1–2aβ chimeras constructs had residues 1–16 and 16–31 from the P2X1 receptor swapped with those from the P2X2 receptor, respectively. P2X1/2eα and P2X1/2eβ chimeras constructs had residues 354–366 and 367–459 from the P2X1 receptor swapped with those from the P2X2 receptor, respectively. B , summary of the effects of cytochalasin D (500 n m , 1 h) treatment on currents mediated by P2X1, P2X2, and P2X1/P2X2 chimeric receptors as fraction of the response to ATP in nontreated cells ( n = 8–19). C , summary of the effects of cytochalasin D (500 n m , 1 h) treatment on currents mediated by P2X1/P2X2 chimeric receptors with modified N terminus ( n = 6–19). *, p < 0.05; ***, p < 0.001.

Article Snippet: Membranes were then processed with the primary anti-P2X1 receptor antibodies (1:1000; Alomone).

Techniques: Construct, Modification

Journal: The Journal of Biological Chemistry

Article Title: Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation *

doi: 10.1074/jbc.M111.253153

Figure Lengend Snippet: Stabilization of actin polymerization prevents disruption of lipid rafts that are essential for P2X1 receptors function. A , representative traces of P2X1 receptor-mediated currents evoked by application of α,β-meATP (10 m m ) for nontreated cells and after incubation with jasplakinolide ( Jasp , 30 n m , 1 h), Mβ-CD (10 m m , 1 h), or jasplakinolide (30 n m ) together with Mβ-CD (10 m m , 1 h) followed by pretreatment with jasplakinolide (30 n m , 1 h). B , summary data of actin polymerization and disruption of lipid rafts on P2X1 receptor-mediated currents. P2X1 receptor currents were unaffected by treatment with jasplakinolide (30 n m , 1 h) which stabilizes polymerized actin cytoskeleton ( n = 9). Mβ-CD (10 m m , 1 h) treatment reduced the peak amplitude of P2X1 receptor currents by >90% ( n = 15). Treatment with jasplakinolide (30 n m , 1 h) together with Mβ-CD (10 m m , 1 h) followed by pretreatment with jasplakinolide (30 n m , 1 h) abolished the effect of Mβ-CD ( n = 13). ***, p < 0.001.

Article Snippet: Membranes were then processed with the primary anti-P2X1 receptor antibodies (1:1000; Alomone).

Techniques: Incubation

Journal: PLoS ONE

Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

doi: 10.1371/journal.pone.0183688

Figure Lengend Snippet: A, Relative expression of P2X 7 mRNA detected by qPCR. B, SDS-PAGE electrophoresis of P2X 7 receptor protein. C, Relative expression of P2X 7 receptor protein; ** P < 0.01, versus Ctrl group; n = 5.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-P2X 7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution).

Techniques: Expressing, SDS Page, Electrophoresis

Journal: PLoS ONE

Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

doi: 10.1371/journal.pone.0183688

Figure Lengend Snippet: A, SDS-PAGE electrophoresis of P2X 7 receptor proteins in primary cultured microglia. B, Relative expression level of P2X 7 receptor protein in primary cultured microglia; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 5.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-P2X 7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution).

Techniques: SDS Page, Electrophoresis, Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

doi: 10.1371/journal.pone.0183688

Figure Lengend Snippet: A, Typical fluorescence image of primary cultured microglia; green signal represents CD11b staining with FITC; red signal indicates P2X 7 staining with TRITC; Merge represents the P2X 7 and CD11b double staining image; arrows represent microglia with co-expression of P2X 7 receptor and CD11b. B, Co-expression values of CD11b and P2X 7 receptor in each group; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 3.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-P2X 7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution).

Techniques: Fluorescence, Cell Culture, Staining, Double Staining, Expressing

Journal: Blood Transfusion

Article Title: Expression and function of purinergic receptors in platelets from apheresis-derived platelet concentrates

doi: 10.2450/2015.0073-15

Figure Lengend Snippet: Impaired calcium flux mediated by P2Y1 and P2X1 receptors in platelets from stored APC.

Article Snippet: The selective P2Y1 receptor agonist [(1R,2R,3S,4R,5S)-4-[6-Amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxy- bicycle [3.1.0] hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS2365), the selective antagonist of P2Y1(1R*,2S*)-4-[2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphornooxy)bicyclo-[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS2500), the agonist of P2X1 receptor α,β-methyleneadenosine 5′-triphosphate trisodium salt (α,β-MeATP), and the potent P2X1 antagonist 4,4′,4″,4‴-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))] tetrakis-1,3-benzenedisulfonic acid, octasodium salt (NF449) were from R&D Systems GmbH (Wiesbaden-Nordenstadt Germany).

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Cryptosporidium parvum -induced neutrophil extracellular traps in neonatal calves is a stage-independent process

doi: 10.3389/fvets.2023.1256726

Figure Lengend Snippet: C. parvum -induced bovine neonatal NET is dependent on purinergic ATP binding. In total, 2 x 10 5 neonatal bovine PMN were pre-treated with the inhibitor NF449 (10μM) for 30min, and C. parvum oocysts were added. Furthermore, there were two control groups: non-stimulated neonatal bovine PMN and neonatal bovine PMN, co-cultured with C. parvum oocysts. The DNA release was significantly inhibited when NF449 was used. p -values were calculated using ANOVA, followed by Dunnett's multiple comparison post-hoc test.

Article Snippet: For P2X1-inhibition assays, freshly isolated neonatal bovine PMN were pre-treated with NF449 (10 μM, Tocris; purinergic receptor antagonist for P2X1) for 30 min and then co-cultured with C. parvum oocysts (1:2 PMN/oocyst ratio, 3 h, 37°C, 5% CO 2 ).

Techniques: Binding Assay, Control, Cell Culture, Comparison