Journal: Frontiers in Pharmacology

Article Title: Differential Expression of the Metabotropic P2Y Receptor Family in the Cortex Following Status Epilepticus and Neuroprotection via P2Y 1 Antagonism in Mice

doi: 10.3389/fphar.2019.01558

Figure Lengend Snippet: Expression profiling of the P2Y receptor family in the cortex following status epilepticus. (A) Photomicrograph (20× lens) showing neuronal damage 24 h following intraamygdala KA-induced status epilepticus in the ipsilateral hippocampus and cortex. Scale bar = 100 μm. (B) Representative Western blot (n = 1 per lane) and corresponding graph showing increased c-Fos expression in the ipsilateral cortex post-status epilepticus (n = 4 per group). (C) Representative Western blots (n = 1 per lane) and corresponding graphs showing the expression of the different P2Y receptor family members P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 12 , P2Y 13 , and P2Y 14 in cortical tissue following status epilepticus. Of note, while P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 are significantly increased post-status epilepticus, no changes could be observed for the remaining P2Y receptors (n = 6 per group). * p < 0.05, ** p < 0.01.

Article Snippet: Membranes were probed with antibodies against P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 12 , P2Y 13 , and P2Y 14 (Alomone Labs, Hadassah Ein Kerem, Jerusalem, Israel), c-Fos (Santa Cruz, Heidelberg, Germany), and β-actin (Sigma-Aldrich, Dublin, Ireland).

Techniques: Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Differential Expression of the Metabotropic P2Y Receptor Family in the Cortex Following Status Epilepticus and Neuroprotection via P2Y 1 Antagonism in Mice

doi: 10.3389/fphar.2019.01558

Figure Lengend Snippet: Cell-specific expression of P2Y 1 post-status epilepticus. (A) Photomicrographs (40× lens) showing co-localization of P2Y 1 (green) with neuronal marker NeuN in cortical tissue (layer V–VI) in control injected-vehicle mice and following status epilepticus (SE) (white arrows). Scale bar = 10 µm (B) Co-localization of P2Y 1 (green) with microglia marker Iba1 (red) 24 h following status epilepticus (SE) in the cortex (white arrows). Scale bar = 10 μm. (C, D) No co-localization of P2Y 1 (green) with the astrocyte markers GFAP and S100β (red). Scale bar = 10 μm. (E) Graph showing strong increase in P2Y 1 -positive microglia 24 h following status epilepticus (n = 3 per group). (F) Enlarged images, outlined by white box, showing co-localization of P2Y 1 (green) on neurons (red) and microglia (red) 24 h post-status epilepticus (SE). Scale bar = 10 μm. (G) Specificity of P2Y 1 -detecting antibody was confirmed using tissue from P2Y 1 knockout mice (KO) 8 h post-status epilepticus. Scale bar = 10 μm. Images are representative image from three animals per experiment. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were probed with antibodies against P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 12 , P2Y 13 , and P2Y 14 (Alomone Labs, Hadassah Ein Kerem, Jerusalem, Israel), c-Fos (Santa Cruz, Heidelberg, Germany), and β-actin (Sigma-Aldrich, Dublin, Ireland).

Techniques: Expressing, Marker, Injection, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: Differential Expression of the Metabotropic P2Y Receptor Family in the Cortex Following Status Epilepticus and Neuroprotection via P2Y 1 Antagonism in Mice

doi: 10.3389/fphar.2019.01558

Figure Lengend Snippet: P2Y 1 antagonism decreases high frequency high amplitude spiking during status epilepticus. (A) Representative EEG traces recorded from the cortex from the time-point of intraamygdala kainic acid (KA) injection until 60 min post-lorazepam (Lz) of mice treated with vehicle (Veh), P2Y 1 agonist MRS2365 (MRS23), and P2Y 1 antagonist MRS2500 (MRS25). Treatment with P2Y 1 -targeting drugs was administered 15 min post-intraamygdala KA injection via i.c.v. Lorazepam (Lz) was administered 40 min following KA injection via i.p. (B) Representative EEG traces showing examples of high frequency and high amplitude (HFHA) spiking taken during the 30 min recording period following drug treatment (see arrows). Mice treated with the P2Y 1 agonist MRS23 showed an increase in the duration of HFHA spiking while mice treated with the P2Y 1 antagonists MRS25 showed a decrease in the duration of HFHA spiking during status epilepticus (n = 7 Veh, 9 MRS23, and 8 MRS25). (C) Representative EEG traces and graph showing slightly increased duration of HFHA spiking during status epilepticus in mice treated with minocycline and P2Y 1 antagonists MRS25 (n = 4 per group). * p < 0.05, ** p < 0.01.

Article Snippet: Membranes were probed with antibodies against P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 12 , P2Y 13 , and P2Y 14 (Alomone Labs, Hadassah Ein Kerem, Jerusalem, Israel), c-Fos (Santa Cruz, Heidelberg, Germany), and β-actin (Sigma-Aldrich, Dublin, Ireland).

Techniques: Injection

Journal: Frontiers in Pharmacology

Article Title: Differential Expression of the Metabotropic P2Y Receptor Family in the Cortex Following Status Epilepticus and Neuroprotection via P2Y 1 Antagonism in Mice

doi: 10.3389/fphar.2019.01558

Figure Lengend Snippet: Decreased status epilepticus-induced cortical damage via P2Y 1 antagonism. (A) Representative images (20× lens) and corresponding graph showing a decrease in neuronal damage in the cortex 24 h post-status epilepticus in mice treated with the P2Y 1 antagonist MRS25 (n = 7 Veh, 9 MRS23, and 8 MRS25). (B) Representative images (20x lens) and corresponding graph showing slightly more Fluoro-Jade B (FjB)-positive cells in ipsilateral cortex 24 h post-status epilepticus in mice treated with both minocycline and the P2Y1 antagonist MRS25 (n = 4/group). ** p < 0.01.

Article Snippet: Membranes were probed with antibodies against P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 12 , P2Y 13 , and P2Y 14 (Alomone Labs, Hadassah Ein Kerem, Jerusalem, Israel), c-Fos (Santa Cruz, Heidelberg, Germany), and β-actin (Sigma-Aldrich, Dublin, Ireland).

Techniques:

Journal: Mediators of Inflammation

Article Title: Sustained Release of Prostaglandin E 2 in Fibroblasts Expressing Ectopically Cyclooxygenase 2 Impairs P2Y-Dependent Ca 2+ -Mobilization

doi: 10.1155/2014/832103

Figure Lengend Snippet: Subcellular distribution of P2Y2, P2Y4, and P2Y6 receptors in MEFs. WT or KI MEFs were cultured and, after changing the medium, were maintained in the absence or presence of 1 μ M DFU for 2 h. Cells were fixed with paraformaldehyde (4%; pH 7.2) and permeabilized with cold methanol at RT. After incubation with anti-P2Y2, anti-P2Y4 and anti-P2Y6 antibodies (1 : 500) overnight at 4°C, cells were visualized by confocal microscopy using a FITC-conjugated secondary Ab (Alexa-Fluor 488, 1 : 1000). Nuclei were stained with Hoechst 33258. Coverslips were mounted in Prolong Gold antifade reagent (Molecular Probes) and the intensity of the fluorescence was measured using Image J software (NIH, Bethesda, MD, USA). Results show the mean + SD of three experiments. ** P < 0.001 versus the same condition in the WT cells.

Article Snippet: Antibodies against P2Y2, P2Y4, and P2Y6 receptors were from Alomone Labs (Jerusalem, Israel) and other antibodies were from Santa Cruz Biotech (Santa Cruz, CA, USA), from Cell Signaling (Danvers, MA, USA), or from the sources previously described [ ].

Techniques: Cell Culture, Incubation, Confocal, Microscopy, Staining, Fluorescence, Software, Immunofluorescence

Journal: Frontiers in Medicine

Article Title: The identification of hub-methylated differentially expressed genes in osteoarthritis patients is based on epigenomic and transcriptomic data

doi: 10.3389/fmed.2023.1219830

Figure Lengend Snippet: Classification of hub genes (A–C) . The difference in HTRA1 ( p = 2.222e-08), P2RY6 ( p = 1.335e-06), and RCAN1 ( p = 0.001) levels could be observed in OA compared with control groups. HTRA1, HtrA serine peptidase 1; P2RY6, pyrimidinergic receptor P2Y6; RCAN1, regulator of calcineurin 1.

Article Snippet: Subsequently, the membranes were exposed to overnight primary antibody incubation at 4°C, including rabbit anti-HTRA1 (1:1000, Proteintech,55,011–1-AP), rabbit anti-P2Y6 (1:1000, Bioss, bs-12075R), mouse anti-GAPDH (1:5000, Proteintech, 60,004–1-Ig), as well as rabbit anti-Calcipressin1 (1:500, Proteintech, 14,869–1-AP).

Techniques:

Journal: Frontiers in Medicine

Article Title: The identification of hub-methylated differentially expressed genes in osteoarthritis patients is based on epigenomic and transcriptomic data

doi: 10.3389/fmed.2023.1219830

Figure Lengend Snippet: GSVA. Pathways enriched by (A) HTRA1; (B) P2RY6; and (C) RCAN1. A positive association is symbolized by a blue band, and a negative association is symbolized by a green band.

Article Snippet: Subsequently, the membranes were exposed to overnight primary antibody incubation at 4°C, including rabbit anti-HTRA1 (1:1000, Proteintech,55,011–1-AP), rabbit anti-P2Y6 (1:1000, Bioss, bs-12075R), mouse anti-GAPDH (1:5000, Proteintech, 60,004–1-Ig), as well as rabbit anti-Calcipressin1 (1:500, Proteintech, 14,869–1-AP).

Techniques:

Journal: Frontiers in Medicine

Article Title: The identification of hub-methylated differentially expressed genes in osteoarthritis patients is based on epigenomic and transcriptomic data

doi: 10.3389/fmed.2023.1219830

Figure Lengend Snippet: By adopting RT-qPCR, the above 3 hub genes were discovered to be differentially expressed in the OA group in relative to the control group. (A) HTRA1, (D) P2RY6, and (G) RCAN1. GAPDH expression was taken as standard for all samples. Means ±SEM ( n = 9) are used to represent values, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group, OA, osteoarthritis. (B,C,E,F,H,I) Quantitative analysis and Western blot assay exhibiting HTRA1, P2RY6, RCAN1 protein levels, and corresponding NCs, with GAPDH being the endogenous reference. In addition, data are indicated by means ±SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the membranes were exposed to overnight primary antibody incubation at 4°C, including rabbit anti-HTRA1 (1:1000, Proteintech,55,011–1-AP), rabbit anti-P2Y6 (1:1000, Bioss, bs-12075R), mouse anti-GAPDH (1:5000, Proteintech, 60,004–1-Ig), as well as rabbit anti-Calcipressin1 (1:500, Proteintech, 14,869–1-AP).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular Pain

Article Title: Role of spinal P2Y 6 and P2Y 11 receptors in neuropathic pain in rats: possible involvement of glial cells

doi: 10.1186/1744-8069-10-29

Figure Lengend Snippet: Intrathecal injection of the selective P2Y 6,11 receptor antagonists reverses tactile allodynia. Time course of the antiallodynic effects of the selective P2Y 6 (MRS2578, panel A ) and P2Y 11 (NF340, panel C ) receptor antagonists in rats submitted to spinal nerve injury. Data are expressed as mean ± S.E.M. for 6 animals. Withdrawal threshold was assessed 14 days after spinal nerve injury. Dose–response curves of the antiallodynic effect of MRS2578 (10–100 μM, panel B ) and NF340 (0.3–30 μM, panel D ). Data are expressed as the 50% threshold withdrawal against time curve (AUC). * Significantly different from the vehicle (Veh) group ( p < 0.05), as determined by analysis of variance followed by the Student-Newman-Keuls test. N: Naive.

Article Snippet: After that, they were incubated overnight at 4°C in 5% non-fat dry milk/PBS containing rabbit anti-P2Y 6 (BML-SA586-0050, 1:100, Enzo Life Sciences, Farmingdale, NY), rabbit anti-P2Y 11 (AB9590, 1:500, Millipore, Billerica, MA), mouse anti-Iba-1 (MABN92, 1:500, Millipore, Bellerica, MA) or rabbit anti-GFAP (AB5804, 1:1000, Millipore, Bellerica, MA) antibodies.

Techniques: Injection

Journal: Molecular Pain

Article Title: Role of spinal P2Y 6 and P2Y 11 receptors in neuropathic pain in rats: possible involvement of glial cells

doi: 10.1186/1744-8069-10-29

Figure Lengend Snippet: Spinal nerve injury enhances the expression of P2Y 6,11 receptors. Western blot analysis of the P2Y 6 (panel A ) and P2Y 11 (panel B ) receptors expression in the ipsilateral dorsal spinal cord obtained from naïve and spinal nerve injured (SNL) rats. The primary antibody for P2Y 6 and P2Y 11 were pre-adsorbed with the corresponding antigenic peptide (+P, panel C ). Data were normalized against β-actin and are expressed as the mean ± S.E.M. of 3 independent rats. *Significantly ( p < 0.05) different from the naïve (N) group, as determined by one-way analysis of variance followed by the Student-Newman-Keuls test.

Article Snippet: After that, they were incubated overnight at 4°C in 5% non-fat dry milk/PBS containing rabbit anti-P2Y 6 (BML-SA586-0050, 1:100, Enzo Life Sciences, Farmingdale, NY), rabbit anti-P2Y 11 (AB9590, 1:500, Millipore, Billerica, MA), mouse anti-Iba-1 (MABN92, 1:500, Millipore, Bellerica, MA) or rabbit anti-GFAP (AB5804, 1:1000, Millipore, Bellerica, MA) antibodies.

Techniques: Expressing, Western Blot

Journal: Molecular Pain

Article Title: Role of spinal P2Y 6 and P2Y 11 receptors in neuropathic pain in rats: possible involvement of glial cells

doi: 10.1186/1744-8069-10-29

Figure Lengend Snippet: P2Y 6,11 receptor antagonists prevent enhanced expression of P2Y 6,11 receptors. Effect of the repeated intrathecal administration of MRS2578 (panel A ) and NF340 (panel B ) in spinal nerve injury-induced increase in P2Y 6 and P2Y 11 expression in the ipsilateral dorsal spinal cord. Data were normalized against β-actin and are expressed as the mean ± S.E.M. of 3 independent rats. * Significantly ( p < 0.05) different from the naïve (N) group and # significantly ( p < 0.05) different from the spinal nerve ligated (SNL) group, as determined by one-way analysis of variance followed by the Student-Newman-Keuls test.

Article Snippet: After that, they were incubated overnight at 4°C in 5% non-fat dry milk/PBS containing rabbit anti-P2Y 6 (BML-SA586-0050, 1:100, Enzo Life Sciences, Farmingdale, NY), rabbit anti-P2Y 11 (AB9590, 1:500, Millipore, Billerica, MA), mouse anti-Iba-1 (MABN92, 1:500, Millipore, Bellerica, MA) or rabbit anti-GFAP (AB5804, 1:1000, Millipore, Bellerica, MA) antibodies.

Techniques: Expressing

Journal: Molecular Pain

Article Title: Role of spinal P2Y 6 and P2Y 11 receptors in neuropathic pain in rats: possible involvement of glial cells

doi: 10.1186/1744-8069-10-29

Figure Lengend Snippet: Spinal nerve injury increases the expression of P2Y 6 and P2Y 11 : Blockade by minocycline or fluorocitrate. Effect of the repeated intrathecal administration of minocycline (Mino, panel A and B ) or fluorocitrate (Fluo, panel C and D ) in spinal nerve injury-induced increased expression of P2Y 6 and P2Y 11 receptors in the ipsilateral dorsal spinal cord. Data were normalized against β-actin and are expressed as the mean ± S.E.M. of 3 independent rats. * Significantly ( p < 0.05) different from the naïve (N) group and # significantly ( p < 0.05) different from the spinal nerve ligated (SNL) group, as determined by one-way analysis of variance followed by the Student-Newman-Keuls test.

Article Snippet: After that, they were incubated overnight at 4°C in 5% non-fat dry milk/PBS containing rabbit anti-P2Y 6 (BML-SA586-0050, 1:100, Enzo Life Sciences, Farmingdale, NY), rabbit anti-P2Y 11 (AB9590, 1:500, Millipore, Billerica, MA), mouse anti-Iba-1 (MABN92, 1:500, Millipore, Bellerica, MA) or rabbit anti-GFAP (AB5804, 1:1000, Millipore, Bellerica, MA) antibodies.

Techniques: Expressing