p2y6  (Alomone Labs)

Journal:

Article Title: Evidence for the Expression of Multiple Uracil Nucleotide-Stimulated P2 Receptors Coupled to Smooth Muscle Contraction in Porcine Isolated Arteries

doi: 10.1038/sj.bjp.0707120

Figure Lengend Snippet: RT-PCR and sequence analysis of porcine P2Y2 , P2Y4 and P2Y6 receptors

Article Snippet: For example, reports describe expression of mRNA for P2Y2 , P2Y4 and P2Y6 receptors in human coronary artery , P2Y2 and P2Y6 receptors in human omental and cerebral arteries ( ) and P2Y2 , P2Y4 and P2Y6 receptors in human placental vasculature ( ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: CNS Neuroscience & Therapeutics

Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

doi: 10.1111/cns.13296

Figure Lengend Snippet: P2Y6 receptor expressed on microglia and dramatically increased within 3 d after tMCAO. A, Western blot and the quantification of P2Y6 receptor at 0 h, 6 h, 12 h, 1, and 3 d after ischemic stroke. B, Quantitative of RT‐PCR analysis for the expression of P2Y6 receptor at 0 h, 6 h, 12 h, 1, and 3 d after tMCAO. C, Representative fluorescence images of P2Y6 receptor in the contralateral and ipsilateral hemispheres of mice after tMCAO. Scale bar = 100 μm. D, Z‐stack for representative fluorescence images of (a) P2Y6 receptor (green) and Iba1 (red), (b) P2Y6 receptor (green) and GFAP (red), and (c) P2Y6 receptor (green) and Tuj 1 (red). Scale bar = 10 μm. Low magnification images are also presented respectively. Scale bar = 50 μm. Data were presented as mean ± SEM (n = 3 per group). * P

Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Fluorescence, Mouse Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

doi: 10.1111/cns.13296

Figure Lengend Snippet: P2Y6 receptor antagonist MRS2578 inhibit MLCK expression in mice at 3 d after tMCAO and cultured microglia. A, (a) Representative fluorescence images of MLCK in Iba‐1 + cultured primary microglia. Scale bar = 25 μm. (b) Quantitative analysis of MLCK in each Iba1 + microglia. n = 4 per group. B, (a) Western blot for the expression of MLCK in the control, LPS+UDP and LPD+UDP+MRS2578 group. (b) Quantification for the intensity ratios of MLCK/β‐actin in the control, LPS+UDP and LPD+UDP+MRS2578 group. C, (a) Western blot for the expression of MLCK in the sham, saline, and MRS2578 group at 3 d after tMCAO. (b) Quantification for the intensity ratios of MLCK/β‐actin in the sham, saline, and MRS2578 group. Data were presented as mean ± SEM. n = 3‐4 per group. * P

Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

Techniques: Expressing, Mouse Assay, Cell Culture, Fluorescence, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

doi: 10.1111/cns.13296

Figure Lengend Snippet: Inhibition of P2Y6 receptor activity reduced the phagocytic function of microglia after tMCAO. A, Representative fluorescence images of Iba1 + microglia (red) and TUNEL + apoptotic cells (green) in the peri‐infarct region of brain slice in the sham (a), saline (b), and MRS2578 (c) group at 3 d after tMCAO. Scale bar = 50 μm. White arrows indicated Iba1 + cells engulfed TUNEL + cells in the saline and MRS2578 group. B, Semiquantitative analysis of Iba1 + microglial phagocytosis for TUNEL + cells. n = 4. C, Representative fluorescence images of Iba1 + microglia (green), lysosome marker LAMP‐2 (purple), and TUNEL + apoptotic cells (red) in the peri‐infarct region of brain slice after tMCAO. 3D scanning with depth indicated Iba1 + cells phagocytosed TUNEL + cells and transferred them into lysosomes. Scale bar = 5 μm. D, Representative fluorescence image of Iba1 + microglia (green), NeuN + neurons (purple), and TUNEL + apoptotic cells (red) in the peri‐infarct region of brain slice after tMCAO. Scale bar = 5 μm. 3D scanning with depth indicated Iba1 + cells engulfed TUNEL + neurons

Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

Techniques: Inhibition, Activity Assay, Fluorescence, TUNEL Assay, Slice Preparation, Marker

Journal: CNS Neuroscience & Therapeutics

Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

doi: 10.1111/cns.13296

Figure Lengend Snippet: P2Y6 receptor‐specific inhibitor MRS2578 blocked the phagocytosis of primary microglia under LPS UDP stimulation. A, Representative images of fluorescent latex beads (yellow) phagocytized by primary microglia under 12 h LPS (200 ng/mL) UDP (100 μmol/L) stimulation in the control, LPS+UDP, 1 μmol/L MRS, 2 μmol/L MRS, and 5 μmol/L MRS group. (a) Control group, only cultured by basic culture medium of microglia, (b) MRS2578‐free group, 200 ng/mL LPS, and 100 μmol/L UDP in basic culture medium of microglia, (c) 1 μmol/L MRS2578 group, 200 ng/mL LPS, 100 μmol/L UDP, and 1 μmol/L MRS2578 in basic culture medium of microglia, (d) 2 μmol/L MRS2578 group, 200 ng/mL LPS, 100 μmol/L UDP, and 2 μmol/L MRS2578 in basic culture medium of microglia, (e) 5 μmol/L MRS2578 group, 200 ng/mL LPS, 100 μmol/L UDP, and 5 μmol/L MRS2578 in basic culture medium of microglia. Scale bar = 10 μm. B, Quantification for the number of beads per microglia under LPS (200 ng/mL) UDP (100 μmol/L) stimulation (n = 3 per group). C, CCK‐8 assay in primary microglia without or with different concentrations MRS2578 under LPS+UDP stimulation for 12 h. D, (a) Representative flow cytometry analysis of microglia phagocytosis. Phagocytosis of fluorescein isothiocyanate (FITC)‐labeled latex beads by CD206 + and CD206 ‐ microglia. (Green, untreated control; Red, LPS+UDP treatment; Blue: LPS+UDP+1 μmol/L MRS2578 treatment); (b) Percentage of FITC + cells in CX 3 CR 1 + cells for total, CD206+, and CD206‐ microglia. E, (a) Representative diagrams of CD206 + /CX 3 CR 1 + cells in the control, LPS+UDP, LPS+UDP+MRS2578 group, (b) Percentage of CD206 + /CX 3 CR 1 + in the control, LPS+UDP, LPS+UDP+MRS2578 group. Data were presented as mean ± SEM. n = 3 per group. * P

Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

Techniques: Cell Culture, CCK-8 Assay, Flow Cytometry, Labeling

Journal: CNS Neuroscience & Therapeutics

Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

doi: 10.1111/cns.13296

Figure Lengend Snippet: Inhibition of P2Y6 receptor activity exacerbated neurological function deficit and brain injury after tMCAO in mice. A, (a) Representative images of cresyl violet staining for brain infarct volume in the sham, saline, and MRS2578 group at 3 d after tMCAO. (b) Quantification of brain infarct volume in the sham, saline, and MRS2578 group at 3 d after tMCAO. (c) Quantification of the percentage of ipsilateral hemisphere volume/contralateral hemisphere volume in the sham, saline, and MRS2578 group at 3 d after tMCAO. B, (d) Representative images of cresyl violet staining for brain atrophy volume in the sham, saline, and MRS2578 group at 14 d after tMCAO. (e) Quantification of brain atrophy volume in the sham, saline, and MRS2578 group at 14 d after tMCAO. (f) Quantification of the percentage of ipsilateral hemisphere volume/contralateral hemisphere volume in the sham, saline, and MRS2578 group at 14 d after tMCAO. C. mNSS in the saline and MRS2578 group at 1, 3, 7, and 14 d after tMCAO. mNSS = modified neurological severity scores. D, Grid walking test in the saline and MRS2578 group at 3, 7, and 14 d after tMCAO. E, Body weight in the saline and MRS2578 group at 1, 3, 7, and 14 d after tMCAO. F, Survival curves of the saline and MRS2578 group within 3 d after tMCAO. Data were presented as mean ± SEM. n = 6‐11 per group. * P

Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

Techniques: Inhibition, Activity Assay, Mouse Assay, Staining, Modification

Journal: CNS Neuroscience & Therapeutics

Article Title: P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

doi: 10.1111/cns.13296

Figure Lengend Snippet: Inhibiting the P2Y6 receptor activity had no effect on the expression of inflammatory cytokines and neutrophil infiltration. A, Quantification of RT‐PCR analysis for the expression of IL‐1α (a), IL‐1β (b), IL‐6 (c), IL‐10 (d), TNF‐α (e), and TGF‐β (f) at 3 d after tMCAO in the sham, saline, and MRS2578 group. The data in sham group were normalized to 1. B, Western blot for the expression of MPO in the brain of sham, saline, and MRS2578 groups at 3 d after tMCAO (g). Quantification of the intensity ratios of MPO/ β‐actin (h) in the sham, saline, and MRS2578 group. The data in the saline group were normalized to 1. C, Quantification of RT‐PCR analysis for the expression of IL‐1α (a), IL‐1β (b), IL‐6 (c), IL‐10(d), TNF‐α (e), and TGF‐β (f) after 1 h OGD and 11 h reoxygenation in the control, OGD and OGD+MRS2578 group. The data in the control group were normalized to 1. Data were presented as mean ± SEM. n = 3‐4 per group. * P

Article Snippet: Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C.

Techniques: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot