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anti rabbit p2y 6 r polyclonal antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti rabbit p2y 6 r polyclonal antibody
    Endothelial <t>P2Y</t> <t>6</t> <t>R</t> activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)
    Anti Rabbit P2y 6 R Polyclonal Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+p2y+6/Anti-LPAR4+Antibody/pmc13013911-54-19-26
    Average 93 stars, based on 1 article reviews
    anti rabbit p2y 6 r polyclonal antibody - by Bioz Stars, 2026-11
    93/100 stars

    Images

    1) Product Images from "Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis"

    Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

    Journal: Purinergic Signalling

    doi: 10.1007/s11302-026-10141-x

    Endothelial P2Y 6 R activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)
    Figure Legend Snippet: Endothelial P2Y 6 R activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)

    Techniques Used: Activation Assay, Control, Infection

    Endothelial P2Y 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)
    Figure Legend Snippet: Endothelial P2Y 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)

    Techniques Used: Immunocytochemistry, Staining, Cell Culture, Control, Infection, Fluorescence

    Endothelial P2Y 6 R downregulation is mediated by proteosome degradation during schistosomiasis. A. Treatment with the ectonucleotidase inhibitor ( ARL67156 100 µM, 24 h) followed by a wash step and UDP 100 µM (5 h); treatment with apyrase 2 U/L (24 h) followed by washing and UDP 100 µM (5 h). B. Treatment with the proteosome inhibitor MG132 (1 µM, 1 h) followed by UDP (100 µM, 5 h). Data were expressed as mean and SEM of n independent cultures in triplicate for each condition. ***p < 0.001, *p < 0.05 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group). ARL = ARL67156
    Figure Legend Snippet: Endothelial P2Y 6 R downregulation is mediated by proteosome degradation during schistosomiasis. A. Treatment with the ectonucleotidase inhibitor ( ARL67156 100 µM, 24 h) followed by a wash step and UDP 100 µM (5 h); treatment with apyrase 2 U/L (24 h) followed by washing and UDP 100 µM (5 h). B. Treatment with the proteosome inhibitor MG132 (1 µM, 1 h) followed by UDP (100 µM, 5 h). Data were expressed as mean and SEM of n independent cultures in triplicate for each condition. ***p < 0.001, *p < 0.05 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group). ARL = ARL67156

    Techniques Used:

    Endothelial P2Y 6 R expression is increased by proteosome inhibition during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from basal (top line) MG132 1 µM, 1 h (middle line) or MG132 1 µM, 6 h (bottom line) using anti-P2Y 6 R antibody (1:500, overnight) followed by Alexa Fluor 488 secondary antibody secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified for all groups and then expressed as arbitrary units (a.u.). C . ROS was quantified colorimetrically using NBT. D . Lipid peroxidation was quantified by the production of malondialdehyde (MDA) in reaction with thiobarbituric acid. Data were expressed as the mean and SEM of n independent cultures in triplicate for each condition. **p < 0.01 ( B , One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group); ***p < 0.001; **p < 0.01 (C, D, unpaired t -test, n = 3 animals for each group). ROS = reactive oxygen species; TBARS = thiobarbituric acid reactive species
    Figure Legend Snippet: Endothelial P2Y 6 R expression is increased by proteosome inhibition during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from basal (top line) MG132 1 µM, 1 h (middle line) or MG132 1 µM, 6 h (bottom line) using anti-P2Y 6 R antibody (1:500, overnight) followed by Alexa Fluor 488 secondary antibody secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified for all groups and then expressed as arbitrary units (a.u.). C . ROS was quantified colorimetrically using NBT. D . Lipid peroxidation was quantified by the production of malondialdehyde (MDA) in reaction with thiobarbituric acid. Data were expressed as the mean and SEM of n independent cultures in triplicate for each condition. **p < 0.01 ( B , One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group); ***p < 0.001; **p < 0.01 (C, D, unpaired t -test, n = 3 animals for each group). ROS = reactive oxygen species; TBARS = thiobarbituric acid reactive species

    Techniques Used: Expressing, Inhibition, Immunocytochemistry, Staining, Cell Culture, Fluorescence

    Proposed mechanism of proteasomal-dependent P2Y 6 receptor downregulation pathway in mesenteric endothelial cells in a preclinical model of intestinal schistosomiasis. The mesenteric endothelial cells primed by infection showed a proteasome-dependent P2Y 6 receptor downregulation under conditions of oxidative stress (1) triggering post-translational modifications protein such as polyubiquitination (2), which targets the receptor to the proteasomal degradation pathway (3–4), ultimately resulting in reduced endothelial P2Y 6 R expression and diminished proinflammatory effect (5)
    Figure Legend Snippet: Proposed mechanism of proteasomal-dependent P2Y 6 receptor downregulation pathway in mesenteric endothelial cells in a preclinical model of intestinal schistosomiasis. The mesenteric endothelial cells primed by infection showed a proteasome-dependent P2Y 6 receptor downregulation under conditions of oxidative stress (1) triggering post-translational modifications protein such as polyubiquitination (2), which targets the receptor to the proteasomal degradation pathway (3–4), ultimately resulting in reduced endothelial P2Y 6 R expression and diminished proinflammatory effect (5)

    Techniques Used: Infection, Expressing

    Related Articles

    Membrane:

    Article Title: Diadenosine tetraphosphate (Ap 4 A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment
    Article Snippet: Homogenates containing 50–100 mg of protein were separated using conventional SDS-polyacrylamide gel electrophoresis in reducing conditions (5 % β-mercaptoethanol; Sigma-Aldrich) and transferred to 0.45 μm pore size polyvinylidenedifluoride membrane (PVDF, Immobilon, Merck Millipore; Darmstadt, Germany). .. The membrane was blocked with a solution of 5 % nonfat milk in TBS-T (Tris buffer saline plus 0.05 % ( v / v ) Tween20) for 1 h at room temperature (RT) and then incubated with the following primary antibodies diluted in blocking solution: anti-P2X 1 (1:500; Alomone Labs (Jerusalem, Israel) cat#APR-001, RRID:AB_2040052), anti-P2X 2 (1:500; Alomone Labs cat#APR-003, RRID:AB_2040054), anti-P2X 4 (1:500; Alomone Labs cat#APR-002, RRID:AB_2040058), anti-P2X 7 (1:500; Alomone Labs cat#APR-008, RRID:AB_2040065), anti-P2Y 1 (1:500; Alomone Labs cat#APR-009, RRID:AB_2040070), anti-P2Y 2 (1:500; Alomone Labs cat#APR-010, RRID:AB_2040078), anti-P2Y 6 (1:500; Alomone Labs cat#APR-011, RRID:AB_2040082), and β-tubulin (1:1000; Sigma-Aldrich Cat# T5293, RRID:AB_477580) as loading control. .. Neuro-2a mouse neuroblastoma cells (CCL-131; ATCC, Manassas, VA, USA) were grown in 100 mm 2 dishes with Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Basel, Switzerland) supplemented with 10 % fetal bovine serum (FBS; Lonza) and 1 % penicillin/streptomycin solution (Gibco, ThermoFisher Scientific, Waltham, MA, USA) at 37 °C and 5 % CO 2 .

    Saline:

    Article Title: Diadenosine tetraphosphate (Ap 4 A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment
    Article Snippet: Homogenates containing 50–100 mg of protein were separated using conventional SDS-polyacrylamide gel electrophoresis in reducing conditions (5 % β-mercaptoethanol; Sigma-Aldrich) and transferred to 0.45 μm pore size polyvinylidenedifluoride membrane (PVDF, Immobilon, Merck Millipore; Darmstadt, Germany). .. The membrane was blocked with a solution of 5 % nonfat milk in TBS-T (Tris buffer saline plus 0.05 % ( v / v ) Tween20) for 1 h at room temperature (RT) and then incubated with the following primary antibodies diluted in blocking solution: anti-P2X 1 (1:500; Alomone Labs (Jerusalem, Israel) cat#APR-001, RRID:AB_2040052), anti-P2X 2 (1:500; Alomone Labs cat#APR-003, RRID:AB_2040054), anti-P2X 4 (1:500; Alomone Labs cat#APR-002, RRID:AB_2040058), anti-P2X 7 (1:500; Alomone Labs cat#APR-008, RRID:AB_2040065), anti-P2Y 1 (1:500; Alomone Labs cat#APR-009, RRID:AB_2040070), anti-P2Y 2 (1:500; Alomone Labs cat#APR-010, RRID:AB_2040078), anti-P2Y 6 (1:500; Alomone Labs cat#APR-011, RRID:AB_2040082), and β-tubulin (1:1000; Sigma-Aldrich Cat# T5293, RRID:AB_477580) as loading control. .. Neuro-2a mouse neuroblastoma cells (CCL-131; ATCC, Manassas, VA, USA) were grown in 100 mm 2 dishes with Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Basel, Switzerland) supplemented with 10 % fetal bovine serum (FBS; Lonza) and 1 % penicillin/streptomycin solution (Gibco, ThermoFisher Scientific, Waltham, MA, USA) at 37 °C and 5 % CO 2 .

    Incubation:

    Article Title: Diadenosine tetraphosphate (Ap 4 A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment
    Article Snippet: Homogenates containing 50–100 mg of protein were separated using conventional SDS-polyacrylamide gel electrophoresis in reducing conditions (5 % β-mercaptoethanol; Sigma-Aldrich) and transferred to 0.45 μm pore size polyvinylidenedifluoride membrane (PVDF, Immobilon, Merck Millipore; Darmstadt, Germany). .. The membrane was blocked with a solution of 5 % nonfat milk in TBS-T (Tris buffer saline plus 0.05 % ( v / v ) Tween20) for 1 h at room temperature (RT) and then incubated with the following primary antibodies diluted in blocking solution: anti-P2X 1 (1:500; Alomone Labs (Jerusalem, Israel) cat#APR-001, RRID:AB_2040052), anti-P2X 2 (1:500; Alomone Labs cat#APR-003, RRID:AB_2040054), anti-P2X 4 (1:500; Alomone Labs cat#APR-002, RRID:AB_2040058), anti-P2X 7 (1:500; Alomone Labs cat#APR-008, RRID:AB_2040065), anti-P2Y 1 (1:500; Alomone Labs cat#APR-009, RRID:AB_2040070), anti-P2Y 2 (1:500; Alomone Labs cat#APR-010, RRID:AB_2040078), anti-P2Y 6 (1:500; Alomone Labs cat#APR-011, RRID:AB_2040082), and β-tubulin (1:1000; Sigma-Aldrich Cat# T5293, RRID:AB_477580) as loading control. .. Neuro-2a mouse neuroblastoma cells (CCL-131; ATCC, Manassas, VA, USA) were grown in 100 mm 2 dishes with Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Basel, Switzerland) supplemented with 10 % fetal bovine serum (FBS; Lonza) and 1 % penicillin/streptomycin solution (Gibco, ThermoFisher Scientific, Waltham, MA, USA) at 37 °C and 5 % CO 2 .

    Article Title: UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells
    Article Snippet: Protein samples were separated on a 10% SDS–PAGE gel and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, United States). .. After blocking in 5% non-fat dry milk buffer, the membranes were incubated overnight at 4°C with rabbit anti-P2Y 2 (1:200; Alomone, Jerusalem, Israel, APR-010), anti-P2Y 4 (1:800; Alomone, APR-006), anti-P2Y 6 (1:600; Alomone, APR-011), TPH1 (1:1000; Novus Biologicals, Littleton, CO, United States, NB110-57629) or anti-mouse β-actin (1:1000; Santa Cruz Biotech, Dallas, TX, United States, sc-47778) in blocking solution. .. Membranes were washed and incubated with the HPR-conjugated secondary anti-rabbit or anti-mouse antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States).

    Blocking Assay:

    Article Title: Diadenosine tetraphosphate (Ap 4 A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment
    Article Snippet: Homogenates containing 50–100 mg of protein were separated using conventional SDS-polyacrylamide gel electrophoresis in reducing conditions (5 % β-mercaptoethanol; Sigma-Aldrich) and transferred to 0.45 μm pore size polyvinylidenedifluoride membrane (PVDF, Immobilon, Merck Millipore; Darmstadt, Germany). .. The membrane was blocked with a solution of 5 % nonfat milk in TBS-T (Tris buffer saline plus 0.05 % ( v / v ) Tween20) for 1 h at room temperature (RT) and then incubated with the following primary antibodies diluted in blocking solution: anti-P2X 1 (1:500; Alomone Labs (Jerusalem, Israel) cat#APR-001, RRID:AB_2040052), anti-P2X 2 (1:500; Alomone Labs cat#APR-003, RRID:AB_2040054), anti-P2X 4 (1:500; Alomone Labs cat#APR-002, RRID:AB_2040058), anti-P2X 7 (1:500; Alomone Labs cat#APR-008, RRID:AB_2040065), anti-P2Y 1 (1:500; Alomone Labs cat#APR-009, RRID:AB_2040070), anti-P2Y 2 (1:500; Alomone Labs cat#APR-010, RRID:AB_2040078), anti-P2Y 6 (1:500; Alomone Labs cat#APR-011, RRID:AB_2040082), and β-tubulin (1:1000; Sigma-Aldrich Cat# T5293, RRID:AB_477580) as loading control. .. Neuro-2a mouse neuroblastoma cells (CCL-131; ATCC, Manassas, VA, USA) were grown in 100 mm 2 dishes with Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Basel, Switzerland) supplemented with 10 % fetal bovine serum (FBS; Lonza) and 1 % penicillin/streptomycin solution (Gibco, ThermoFisher Scientific, Waltham, MA, USA) at 37 °C and 5 % CO 2 .

    Article Title: Changes in P2Y 6 receptor‐mediated vasoreactivity following focal and global ischemia
    Article Snippet: .. The cell suspensions were stained overnight at +4°C temperature with primary goat anti‐SM22α (1:100, Abcam) and primary rabbit anti‐P2Y 6 (1:100, Alomone), respectively or goat isotope control IgG (Abcam), rabbit isotope control IgG (Abcam) in blocking buffer (PBS + 5% normal donkey serum) protected from light with rocking, respectively. ..

    Article Title: UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells
    Article Snippet: Protein samples were separated on a 10% SDS–PAGE gel and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, United States). .. After blocking in 5% non-fat dry milk buffer, the membranes were incubated overnight at 4°C with rabbit anti-P2Y 2 (1:200; Alomone, Jerusalem, Israel, APR-010), anti-P2Y 4 (1:800; Alomone, APR-006), anti-P2Y 6 (1:600; Alomone, APR-011), TPH1 (1:1000; Novus Biologicals, Littleton, CO, United States, NB110-57629) or anti-mouse β-actin (1:1000; Santa Cruz Biotech, Dallas, TX, United States, sc-47778) in blocking solution. .. Membranes were washed and incubated with the HPR-conjugated secondary anti-rabbit or anti-mouse antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States).

    Control:

    Article Title: Diadenosine tetraphosphate (Ap 4 A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment
    Article Snippet: Homogenates containing 50–100 mg of protein were separated using conventional SDS-polyacrylamide gel electrophoresis in reducing conditions (5 % β-mercaptoethanol; Sigma-Aldrich) and transferred to 0.45 μm pore size polyvinylidenedifluoride membrane (PVDF, Immobilon, Merck Millipore; Darmstadt, Germany). .. The membrane was blocked with a solution of 5 % nonfat milk in TBS-T (Tris buffer saline plus 0.05 % ( v / v ) Tween20) for 1 h at room temperature (RT) and then incubated with the following primary antibodies diluted in blocking solution: anti-P2X 1 (1:500; Alomone Labs (Jerusalem, Israel) cat#APR-001, RRID:AB_2040052), anti-P2X 2 (1:500; Alomone Labs cat#APR-003, RRID:AB_2040054), anti-P2X 4 (1:500; Alomone Labs cat#APR-002, RRID:AB_2040058), anti-P2X 7 (1:500; Alomone Labs cat#APR-008, RRID:AB_2040065), anti-P2Y 1 (1:500; Alomone Labs cat#APR-009, RRID:AB_2040070), anti-P2Y 2 (1:500; Alomone Labs cat#APR-010, RRID:AB_2040078), anti-P2Y 6 (1:500; Alomone Labs cat#APR-011, RRID:AB_2040082), and β-tubulin (1:1000; Sigma-Aldrich Cat# T5293, RRID:AB_477580) as loading control. .. Neuro-2a mouse neuroblastoma cells (CCL-131; ATCC, Manassas, VA, USA) were grown in 100 mm 2 dishes with Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Basel, Switzerland) supplemented with 10 % fetal bovine serum (FBS; Lonza) and 1 % penicillin/streptomycin solution (Gibco, ThermoFisher Scientific, Waltham, MA, USA) at 37 °C and 5 % CO 2 .

    Article Title: Changes in P2Y 6 receptor‐mediated vasoreactivity following focal and global ischemia
    Article Snippet: .. The cell suspensions were stained overnight at +4°C temperature with primary goat anti‐SM22α (1:100, Abcam) and primary rabbit anti‐P2Y 6 (1:100, Alomone), respectively or goat isotope control IgG (Abcam), rabbit isotope control IgG (Abcam) in blocking buffer (PBS + 5% normal donkey serum) protected from light with rocking, respectively. ..

    Staining:

    Article Title: Changes in P2Y 6 receptor‐mediated vasoreactivity following focal and global ischemia
    Article Snippet: .. The cell suspensions were stained overnight at +4°C temperature with primary goat anti‐SM22α (1:100, Abcam) and primary rabbit anti‐P2Y 6 (1:100, Alomone), respectively or goat isotope control IgG (Abcam), rabbit isotope control IgG (Abcam) in blocking buffer (PBS + 5% normal donkey serum) protected from light with rocking, respectively. ..

    Activity Assay:

    Article Title: P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation
    Article Snippet: .. Drugs and antibodies The following antibodies and drugs were used: goat anti-mouse IgG conjugated to Alexa Fluor 488 from Invitrogen (Barcelona, Spain); rabbit polyclonal anti-P2Y 1 and anti-P2Y 6 from Alomone Laboratories (Jerusalem, Israel); mouse monoclonal anti-CD11b, rabbit polyclonal anti-actin and goat anti-rabbit IgG conjugated to horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-P2Y 12 , rabbit and mouse anti-glial fibrillary acidic protein (anti-GFAP), goat anti-rabbit IgG conjugated to crystalline tetramethylrodamine isothiocyanate (TRITC), adenosine, adenosine-5’-monophosphate (AMP), adenosine-5’-diphosphate tetrasodium (ADP), adenosine 5’- O -(3-thio)-diphosphate tetralithium (ADPβS), adenosine-5’-triphosphate disodium (ATP), adenosine 5’- O -(3-thio)-triphosphate tetralithium (ATPγS), cytosine β- d -arabinofuranoside (Ara-C), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1 H-benzimidazole trihydrochloride hydrate (Hoechst 33258), hypoxanthine, inosine, l -leucine methyl ester hydrochloride (LME), penicillin, streptomycin, uracil, uridine, uridine-5’-monophosphate disodium (UMP), uridine-5’-diphosphate sodium (UDP), uridine-5’-triphosphate trisodium (UTP) and uridine 5′-diphosphoglucose disodium (UDP-glucose) and methyl green from Sigma-Aldrich (Sintra, Portugal); methyl -[ 3 H]-thymidine (specific activity 80–86 Ci.mmol -1 ) and enhanced chemiluminescence Western blotting system from Amersham Biosciences (Lisbon, Portugal); Sulfo-NHS-SS-Biotin and Ultralink Immobilized Neutravidin from Pierce (Rockford, IL, USA). ..

    Article Title: P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation
    Article Snippet: .. The following antibodies and drugs were used: goat anti-mouse IgG conjugated to Alexa Fluor 488 from Invitrogen (Barcelona, Spain); rabbit polyclonal anti-P2Y 1 and anti-P2Y 6 from Alomone Laboratories (Jerusalem, Israel); mouse monoclonal anti-CD11b, rabbit polyclonal anti-actin and goat anti-rabbit IgG conjugated to horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-P2Y 12 , rabbit and mouse anti-glial fibrillary acidic protein (anti-GFAP), goat anti-rabbit IgG conjugated to crystalline tetramethylrodamine isothiocyanate (TRITC), adenosine, adenosine-5’-monophosphate (AMP), adenosine-5’-diphosphate tetrasodium (ADP), adenosine 5’- O -(3-thio)-diphosphate tetralithium (ADPβS), adenosine-5’-triphosphate disodium (ATP), adenosine 5’- O -(3-thio)-triphosphate tetralithium (ATPγS), cytosine β- d -arabinofuranoside (Ara-C), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1 H-benzimidazole trihydrochloride hydrate (Hoechst 33258), hypoxanthine, inosine, l -leucine methyl ester hydrochloride (LME), penicillin, streptomycin, uracil, uridine, uridine-5’-monophosphate disodium (UMP), uridine-5’-diphosphate sodium (UDP), uridine-5’-triphosphate trisodium (UTP) and uridine 5′-diphosphoglucose disodium (UDP-glucose) and methyl green from Sigma-Aldrich (Sintra, Portugal); methyl -[ 3 H]-thymidine (specific activity 80–86 Ci.mmol -1 ) and enhanced chemiluminescence Western blotting system from Amersham Biosciences (Lisbon, Portugal); Sulfo-NHS-SS-Biotin and Ultralink Immobilized Neutravidin from Pierce (Rockford, IL, USA). ..

    Western Blot:

    Article Title: P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation
    Article Snippet: .. Drugs and antibodies The following antibodies and drugs were used: goat anti-mouse IgG conjugated to Alexa Fluor 488 from Invitrogen (Barcelona, Spain); rabbit polyclonal anti-P2Y 1 and anti-P2Y 6 from Alomone Laboratories (Jerusalem, Israel); mouse monoclonal anti-CD11b, rabbit polyclonal anti-actin and goat anti-rabbit IgG conjugated to horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-P2Y 12 , rabbit and mouse anti-glial fibrillary acidic protein (anti-GFAP), goat anti-rabbit IgG conjugated to crystalline tetramethylrodamine isothiocyanate (TRITC), adenosine, adenosine-5’-monophosphate (AMP), adenosine-5’-diphosphate tetrasodium (ADP), adenosine 5’- O -(3-thio)-diphosphate tetralithium (ADPβS), adenosine-5’-triphosphate disodium (ATP), adenosine 5’- O -(3-thio)-triphosphate tetralithium (ATPγS), cytosine β- d -arabinofuranoside (Ara-C), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1 H-benzimidazole trihydrochloride hydrate (Hoechst 33258), hypoxanthine, inosine, l -leucine methyl ester hydrochloride (LME), penicillin, streptomycin, uracil, uridine, uridine-5’-monophosphate disodium (UMP), uridine-5’-diphosphate sodium (UDP), uridine-5’-triphosphate trisodium (UTP) and uridine 5′-diphosphoglucose disodium (UDP-glucose) and methyl green from Sigma-Aldrich (Sintra, Portugal); methyl -[ 3 H]-thymidine (specific activity 80–86 Ci.mmol -1 ) and enhanced chemiluminescence Western blotting system from Amersham Biosciences (Lisbon, Portugal); Sulfo-NHS-SS-Biotin and Ultralink Immobilized Neutravidin from Pierce (Rockford, IL, USA). ..

    Article Title: P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation
    Article Snippet: .. The following antibodies and drugs were used: goat anti-mouse IgG conjugated to Alexa Fluor 488 from Invitrogen (Barcelona, Spain); rabbit polyclonal anti-P2Y 1 and anti-P2Y 6 from Alomone Laboratories (Jerusalem, Israel); mouse monoclonal anti-CD11b, rabbit polyclonal anti-actin and goat anti-rabbit IgG conjugated to horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-P2Y 12 , rabbit and mouse anti-glial fibrillary acidic protein (anti-GFAP), goat anti-rabbit IgG conjugated to crystalline tetramethylrodamine isothiocyanate (TRITC), adenosine, adenosine-5’-monophosphate (AMP), adenosine-5’-diphosphate tetrasodium (ADP), adenosine 5’- O -(3-thio)-diphosphate tetralithium (ADPβS), adenosine-5’-triphosphate disodium (ATP), adenosine 5’- O -(3-thio)-triphosphate tetralithium (ATPγS), cytosine β- d -arabinofuranoside (Ara-C), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1 H-benzimidazole trihydrochloride hydrate (Hoechst 33258), hypoxanthine, inosine, l -leucine methyl ester hydrochloride (LME), penicillin, streptomycin, uracil, uridine, uridine-5’-monophosphate disodium (UMP), uridine-5’-diphosphate sodium (UDP), uridine-5’-triphosphate trisodium (UTP) and uridine 5′-diphosphoglucose disodium (UDP-glucose) and methyl green from Sigma-Aldrich (Sintra, Portugal); methyl -[ 3 H]-thymidine (specific activity 80–86 Ci.mmol -1 ) and enhanced chemiluminescence Western blotting system from Amersham Biosciences (Lisbon, Portugal); Sulfo-NHS-SS-Biotin and Ultralink Immobilized Neutravidin from Pierce (Rockford, IL, USA). ..



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    93
    Alomone Labs anti rabbit p2y 6 r polyclonal antibody
    Endothelial <t>P2Y</t> <t>6</t> <t>R</t> activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)
    Anti Rabbit P2y 6 R Polyclonal Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    primary antibody anti p2y 6 r  (Alomone Labs)
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    Endothelial <t>P2Y</t> 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)
    Primary Antibody Anti P2y 6 R, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p2y 6  (Alomone Labs)
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    Mechanical stimulation (SS) increases the amount osteogenic transcription factors, osterix and osteopontin (OPN), in Pm BM-MSCs via a mechanism dependent on tonic P2X7 and <t>P2Y</t> 6 receptors activation. BM-MSCs grown in an osteoblast-inducing medium for 21 days, either in the absence or presence of shear-stress mechanical stimulation. Mechanically-stimulated Pm BM-MSCs were also tested in the absence (SS group) and in the presence of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Panels Ai and Bi , show typical immunoblots stained for osterix (45 kDa) and OPN (60 − 27 kDa) transcription factors of cultured BM-MSCs obtained from Pm women with 70 and 57 years-old, respectively; β-Tubulin (55 kDa) was used as a house-keeping gene product standard for normalization purposes. Uncropped full-length gel blots may be found in Supplementary Fig. . Panels Aii and Bii show the relative amounts of indicated transcription factors at culture day 21. The results are in percentage of non-stimulated cells (CTR) of the same individual, considered as 100% for normalization purposes (dashed horizontal line). The relative protein amounts in non-stimulated cells were 0.27 ± 0.04 for osterix/β-Tubulin and 1.8 ± 0.4 for OPN/β-Tubulin. Boxes and whiskers represent pooled data from 4 to 6 Pm women (66 ± 4 years old); a P <0.05 (non-parametric Wilcoxon matched-pairs signed rank test, considering 100% for the CTR situation) represents significant differences. Panel Biii shows the relative protein amount of each OPN cleavage products (60, 45, 37 and 27 kDa) obtained in all tested subgroups
    P2y 6, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p2y 6 r  (Alomone Labs)
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    Mechanical stimulation (SS) increases the amount osteogenic transcription factors, osterix and osteopontin (OPN), in Pm BM-MSCs via a mechanism dependent on tonic P2X7 and <t>P2Y</t> 6 receptors activation. BM-MSCs grown in an osteoblast-inducing medium for 21 days, either in the absence or presence of shear-stress mechanical stimulation. Mechanically-stimulated Pm BM-MSCs were also tested in the absence (SS group) and in the presence of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Panels Ai and Bi , show typical immunoblots stained for osterix (45 kDa) and OPN (60 − 27 kDa) transcription factors of cultured BM-MSCs obtained from Pm women with 70 and 57 years-old, respectively; β-Tubulin (55 kDa) was used as a house-keeping gene product standard for normalization purposes. Uncropped full-length gel blots may be found in Supplementary Fig. . Panels Aii and Bii show the relative amounts of indicated transcription factors at culture day 21. The results are in percentage of non-stimulated cells (CTR) of the same individual, considered as 100% for normalization purposes (dashed horizontal line). The relative protein amounts in non-stimulated cells were 0.27 ± 0.04 for osterix/β-Tubulin and 1.8 ± 0.4 for OPN/β-Tubulin. Boxes and whiskers represent pooled data from 4 to 6 Pm women (66 ± 4 years old); a P <0.05 (non-parametric Wilcoxon matched-pairs signed rank test, considering 100% for the CTR situation) represents significant differences. Panel Biii shows the relative protein amount of each OPN cleavage products (60, 45, 37 and 27 kDa) obtained in all tested subgroups
    P2y 6 R, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti p2y 6 r antibodies  (Alomone Labs)
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    Alomone Labs rabbit anti p2y 6 r antibodies
    Mechanical stimulation (SS) increases the amount osteogenic transcription factors, osterix and osteopontin (OPN), in Pm BM-MSCs via a mechanism dependent on tonic P2X7 and <t>P2Y</t> 6 receptors activation. BM-MSCs grown in an osteoblast-inducing medium for 21 days, either in the absence or presence of shear-stress mechanical stimulation. Mechanically-stimulated Pm BM-MSCs were also tested in the absence (SS group) and in the presence of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Panels Ai and Bi , show typical immunoblots stained for osterix (45 kDa) and OPN (60 − 27 kDa) transcription factors of cultured BM-MSCs obtained from Pm women with 70 and 57 years-old, respectively; β-Tubulin (55 kDa) was used as a house-keeping gene product standard for normalization purposes. Uncropped full-length gel blots may be found in Supplementary Fig. . Panels Aii and Bii show the relative amounts of indicated transcription factors at culture day 21. The results are in percentage of non-stimulated cells (CTR) of the same individual, considered as 100% for normalization purposes (dashed horizontal line). The relative protein amounts in non-stimulated cells were 0.27 ± 0.04 for osterix/β-Tubulin and 1.8 ± 0.4 for OPN/β-Tubulin. Boxes and whiskers represent pooled data from 4 to 6 Pm women (66 ± 4 years old); a P <0.05 (non-parametric Wilcoxon matched-pairs signed rank test, considering 100% for the CTR situation) represents significant differences. Panel Biii shows the relative protein amount of each OPN cleavage products (60, 45, 37 and 27 kDa) obtained in all tested subgroups
    Rabbit Anti P2y 6 R Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti-p2y 6  (Enzo Biochem)
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    Enzo Biochem rabbit anti-p2y 6
    Intrathecal injection of the selective <t>P2Y</t> 6,11 receptor antagonists reverses tactile allodynia. Time course of the antiallodynic effects of the selective <t>P2Y</t> <t>6</t> (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.
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    anti p2y 6  (Alomone Labs)
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    Alomone Labs anti p2y 6
    Confocal microscope images demonstrating expression of <t>P2Y</t> <t>6</t> (FITC, green) in cross‐sections of middle cerebral arteries. DAPI (4′,6‐Diamidine‐2′‐phenylindole dihydrochloride, a blue nucleus stain, has been added. The tMCAO, Sham, and SAH images were generated by superimposing a DAPI stained image on a FITC stained image. The negative control demonstrates the lack of unspecific antibody binding and the autofluorescence of the lamina elastica interna which separates the intima from the underlying layer of smooth muscle cells
    Anti P2y 6, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti-p2y6 receptor antibody  (Alomone Labs)
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    Alomone Labs anti-p2y6 receptor antibody
    Confocal microscope images demonstrating expression of <t>P2Y</t> <t>6</t> (FITC, green) in cross‐sections of middle cerebral arteries. DAPI (4′,6‐Diamidine‐2′‐phenylindole dihydrochloride, a blue nucleus stain, has been added. The tMCAO, Sham, and SAH images were generated by superimposing a DAPI stained image on a FITC stained image. The negative control demonstrates the lack of unspecific antibody binding and the autofluorescence of the lamina elastica interna which separates the intima from the underlying layer of smooth muscle cells
    Anti P2y6 Receptor Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Endothelial P2Y 6 R activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)

    Journal: Purinergic Signalling

    Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

    doi: 10.1007/s11302-026-10141-x

    Figure Lengend Snippet: Endothelial P2Y 6 R activation failed to increase monocyte adhesion during schistosomiasis. A. Monocyte adhesion induced by UDP 100 µM was blocked by the selective P2Y 6 R antagonist (MRS2578 1 µM) in the control group (white bars). B. UDP 100 µM stimulus did not increase monocyte adhesion in the infected group (gray bars). ARL67156 100 µM = ectonucleotidase inhibitor (added 30 min before). Data were expressed as mean and SEM of n independent experiments performed in triplicate for each condition. ***p < 0.001 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 4 animals for each group)

    Article Snippet: After that, cells were washed twice for 5 min with Triton X-100 0.2% diluted in PBS and incubated with anti-rabbit P2Y 6 R polyclonal antibody (1:500, Alomone Labs, Israel) overnight.

    Techniques: Activation Assay, Control, Infection

    Endothelial P2Y 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)

    Journal: Purinergic Signalling

    Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

    doi: 10.1007/s11302-026-10141-x

    Figure Lengend Snippet: Endothelial P2Y 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)

    Article Snippet: After that, cells were washed twice for 5 min with Triton X-100 0.2% diluted in PBS and incubated with anti-rabbit P2Y 6 R polyclonal antibody (1:500, Alomone Labs, Israel) overnight.

    Techniques: Immunocytochemistry, Staining, Cell Culture, Control, Infection, Fluorescence

    Endothelial P2Y 6 R downregulation is mediated by proteosome degradation during schistosomiasis. A. Treatment with the ectonucleotidase inhibitor ( ARL67156 100 µM, 24 h) followed by a wash step and UDP 100 µM (5 h); treatment with apyrase 2 U/L (24 h) followed by washing and UDP 100 µM (5 h). B. Treatment with the proteosome inhibitor MG132 (1 µM, 1 h) followed by UDP (100 µM, 5 h). Data were expressed as mean and SEM of n independent cultures in triplicate for each condition. ***p < 0.001, *p < 0.05 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group). ARL = ARL67156

    Journal: Purinergic Signalling

    Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

    doi: 10.1007/s11302-026-10141-x

    Figure Lengend Snippet: Endothelial P2Y 6 R downregulation is mediated by proteosome degradation during schistosomiasis. A. Treatment with the ectonucleotidase inhibitor ( ARL67156 100 µM, 24 h) followed by a wash step and UDP 100 µM (5 h); treatment with apyrase 2 U/L (24 h) followed by washing and UDP 100 µM (5 h). B. Treatment with the proteosome inhibitor MG132 (1 µM, 1 h) followed by UDP (100 µM, 5 h). Data were expressed as mean and SEM of n independent cultures in triplicate for each condition. ***p < 0.001, *p < 0.05 (One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group). ARL = ARL67156

    Article Snippet: After that, cells were washed twice for 5 min with Triton X-100 0.2% diluted in PBS and incubated with anti-rabbit P2Y 6 R polyclonal antibody (1:500, Alomone Labs, Israel) overnight.

    Techniques:

    Endothelial P2Y 6 R expression is increased by proteosome inhibition during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from basal (top line) MG132 1 µM, 1 h (middle line) or MG132 1 µM, 6 h (bottom line) using anti-P2Y 6 R antibody (1:500, overnight) followed by Alexa Fluor 488 secondary antibody secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified for all groups and then expressed as arbitrary units (a.u.). C . ROS was quantified colorimetrically using NBT. D . Lipid peroxidation was quantified by the production of malondialdehyde (MDA) in reaction with thiobarbituric acid. Data were expressed as the mean and SEM of n independent cultures in triplicate for each condition. **p < 0.01 ( B , One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group); ***p < 0.001; **p < 0.01 (C, D, unpaired t -test, n = 3 animals for each group). ROS = reactive oxygen species; TBARS = thiobarbituric acid reactive species

    Journal: Purinergic Signalling

    Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

    doi: 10.1007/s11302-026-10141-x

    Figure Lengend Snippet: Endothelial P2Y 6 R expression is increased by proteosome inhibition during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from basal (top line) MG132 1 µM, 1 h (middle line) or MG132 1 µM, 6 h (bottom line) using anti-P2Y 6 R antibody (1:500, overnight) followed by Alexa Fluor 488 secondary antibody secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified for all groups and then expressed as arbitrary units (a.u.). C . ROS was quantified colorimetrically using NBT. D . Lipid peroxidation was quantified by the production of malondialdehyde (MDA) in reaction with thiobarbituric acid. Data were expressed as the mean and SEM of n independent cultures in triplicate for each condition. **p < 0.01 ( B , One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group); ***p < 0.001; **p < 0.01 (C, D, unpaired t -test, n = 3 animals for each group). ROS = reactive oxygen species; TBARS = thiobarbituric acid reactive species

    Article Snippet: After that, cells were washed twice for 5 min with Triton X-100 0.2% diluted in PBS and incubated with anti-rabbit P2Y 6 R polyclonal antibody (1:500, Alomone Labs, Israel) overnight.

    Techniques: Expressing, Inhibition, Immunocytochemistry, Staining, Cell Culture, Fluorescence

    Proposed mechanism of proteasomal-dependent P2Y 6 receptor downregulation pathway in mesenteric endothelial cells in a preclinical model of intestinal schistosomiasis. The mesenteric endothelial cells primed by infection showed a proteasome-dependent P2Y 6 receptor downregulation under conditions of oxidative stress (1) triggering post-translational modifications protein such as polyubiquitination (2), which targets the receptor to the proteasomal degradation pathway (3–4), ultimately resulting in reduced endothelial P2Y 6 R expression and diminished proinflammatory effect (5)

    Journal: Purinergic Signalling

    Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

    doi: 10.1007/s11302-026-10141-x

    Figure Lengend Snippet: Proposed mechanism of proteasomal-dependent P2Y 6 receptor downregulation pathway in mesenteric endothelial cells in a preclinical model of intestinal schistosomiasis. The mesenteric endothelial cells primed by infection showed a proteasome-dependent P2Y 6 receptor downregulation under conditions of oxidative stress (1) triggering post-translational modifications protein such as polyubiquitination (2), which targets the receptor to the proteasomal degradation pathway (3–4), ultimately resulting in reduced endothelial P2Y 6 R expression and diminished proinflammatory effect (5)

    Article Snippet: After that, cells were washed twice for 5 min with Triton X-100 0.2% diluted in PBS and incubated with anti-rabbit P2Y 6 R polyclonal antibody (1:500, Alomone Labs, Israel) overnight.

    Techniques: Infection, Expressing

    Endothelial P2Y 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)

    Journal: Purinergic Signalling

    Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

    doi: 10.1007/s11302-026-10141-x

    Figure Lengend Snippet: Endothelial P2Y 6 R mRNA and protein levels are reduced during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from control (top line) and infected (bottom line) groups using anti-P2Y 6 R antibody (1:500, overnight), followed by Alexa Fluor 488 secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified and then expressed as arbitrary units (a.u.). P2Y 6 R mRNA levels were lower in the infected (gray bars) than in the control (white bars) group. C. The P2Y 6 R mRNA levels were normalized by the endogenous gene β-actin. Data were expressed as mean and SEM of n independent cultures performed with different animals for each condition. ***p < 0.001, **p < 0.01 (unpaired t -test, n = 3–4 animals for each group)

    Article Snippet: Primary antibody anti-P2Y 6 R (#APR-011) was obtained from Alomone Labs.

    Techniques: Immunocytochemistry, Staining, Cell Culture, Control, Infection, Fluorescence, Immunofluorescence, Real-time Polymerase Chain Reaction

    Endothelial P2Y 6 R expression is increased by proteosome inhibition during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from basal (top line) MG132 1 µM, 1 h (middle line) or MG132 1 µM, 6 h (bottom line) using anti-P2Y 6 R antibody (1:500, overnight) followed by Alexa Fluor 488 secondary antibody secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified for all groups and then expressed as arbitrary units (a.u.). C . ROS was quantified colorimetrically using NBT. D . Lipid peroxidation was quantified by the production of malondialdehyde (MDA) in reaction with thiobarbituric acid. Data were expressed as the mean and SEM of n independent cultures in triplicate for each condition. **p < 0.01 ( B , One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group); ***p < 0.001; **p < 0.01 (C, D, unpaired t -test, n = 3 animals for each group). ROS = reactive oxygen species; TBARS = thiobarbituric acid reactive species

    Journal: Purinergic Signalling

    Article Title: Proteasomal-dependent endothelial P2Y 6 receptor downregulation as an adaptive mechanism limiting monocyte adhesion during intestinal schistosomiasis

    doi: 10.1007/s11302-026-10141-x

    Figure Lengend Snippet: Endothelial P2Y 6 R expression is increased by proteosome inhibition during schistosomiasis. A. Immunocytochemistry staining of cultured mesenteric endothelial cells from basal (top line) MG132 1 µM, 1 h (middle line) or MG132 1 µM, 6 h (bottom line) using anti-P2Y 6 R antibody (1:500, overnight) followed by Alexa Fluor 488 secondary antibody secondary antibody (1:300, 1 h, green) and nuclear fluorescence using DAPI (blue) (scale bar = 20 μm; × 400) (see methods). B. The fluorescence intensity was quantified for all groups and then expressed as arbitrary units (a.u.). C . ROS was quantified colorimetrically using NBT. D . Lipid peroxidation was quantified by the production of malondialdehyde (MDA) in reaction with thiobarbituric acid. Data were expressed as the mean and SEM of n independent cultures in triplicate for each condition. **p < 0.01 ( B , One-way ANOVA followed by Tukey’s multiple comparisons test, n = 3 animals for each group); ***p < 0.001; **p < 0.01 (C, D, unpaired t -test, n = 3 animals for each group). ROS = reactive oxygen species; TBARS = thiobarbituric acid reactive species

    Article Snippet: Primary antibody anti-P2Y 6 R (#APR-011) was obtained from Alomone Labs.

    Techniques: Expressing, Inhibition, Immunocytochemistry, Staining, Cell Culture, Fluorescence, Immunofluorescence

    Mechanical stimulation (SS) increases the amount osteogenic transcription factors, osterix and osteopontin (OPN), in Pm BM-MSCs via a mechanism dependent on tonic P2X7 and P2Y 6 receptors activation. BM-MSCs grown in an osteoblast-inducing medium for 21 days, either in the absence or presence of shear-stress mechanical stimulation. Mechanically-stimulated Pm BM-MSCs were also tested in the absence (SS group) and in the presence of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Panels Ai and Bi , show typical immunoblots stained for osterix (45 kDa) and OPN (60 − 27 kDa) transcription factors of cultured BM-MSCs obtained from Pm women with 70 and 57 years-old, respectively; β-Tubulin (55 kDa) was used as a house-keeping gene product standard for normalization purposes. Uncropped full-length gel blots may be found in Supplementary Fig. . Panels Aii and Bii show the relative amounts of indicated transcription factors at culture day 21. The results are in percentage of non-stimulated cells (CTR) of the same individual, considered as 100% for normalization purposes (dashed horizontal line). The relative protein amounts in non-stimulated cells were 0.27 ± 0.04 for osterix/β-Tubulin and 1.8 ± 0.4 for OPN/β-Tubulin. Boxes and whiskers represent pooled data from 4 to 6 Pm women (66 ± 4 years old); a P <0.05 (non-parametric Wilcoxon matched-pairs signed rank test, considering 100% for the CTR situation) represents significant differences. Panel Biii shows the relative protein amount of each OPN cleavage products (60, 45, 37 and 27 kDa) obtained in all tested subgroups

    Journal: Stem Cell Research & Therapy

    Article Title: Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women

    doi: 10.1186/s13287-024-03775-4

    Figure Lengend Snippet: Mechanical stimulation (SS) increases the amount osteogenic transcription factors, osterix and osteopontin (OPN), in Pm BM-MSCs via a mechanism dependent on tonic P2X7 and P2Y 6 receptors activation. BM-MSCs grown in an osteoblast-inducing medium for 21 days, either in the absence or presence of shear-stress mechanical stimulation. Mechanically-stimulated Pm BM-MSCs were also tested in the absence (SS group) and in the presence of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Panels Ai and Bi , show typical immunoblots stained for osterix (45 kDa) and OPN (60 − 27 kDa) transcription factors of cultured BM-MSCs obtained from Pm women with 70 and 57 years-old, respectively; β-Tubulin (55 kDa) was used as a house-keeping gene product standard for normalization purposes. Uncropped full-length gel blots may be found in Supplementary Fig. . Panels Aii and Bii show the relative amounts of indicated transcription factors at culture day 21. The results are in percentage of non-stimulated cells (CTR) of the same individual, considered as 100% for normalization purposes (dashed horizontal line). The relative protein amounts in non-stimulated cells were 0.27 ± 0.04 for osterix/β-Tubulin and 1.8 ± 0.4 for OPN/β-Tubulin. Boxes and whiskers represent pooled data from 4 to 6 Pm women (66 ± 4 years old); a P <0.05 (non-parametric Wilcoxon matched-pairs signed rank test, considering 100% for the CTR situation) represents significant differences. Panel Biii shows the relative protein amount of each OPN cleavage products (60, 45, 37 and 27 kDa) obtained in all tested subgroups

    Article Snippet: Anti-human P2X7 (anti-rabbit, Cat. No. APR-008, RRID: AB_2040065) and P2Y 6 (anti-rabbit, Cat. No. APR-011, RRID: AB_2040082) were purchased from Alomone (Jerusalem, Israel); anti-human Osterix (anti-rabbit, Cat No. ab22552, RRID: AB_2194492), anti-human β-Tubulin (anti-rabbit, Cat. No. ab6046, RRID: AB_2210370) and horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit, Cat. No. ab7083, RRID: AB_955416 and anti-mouse, Cat No. ab6820, RRID: AB_955438) were purchased from Abcam (Cambridge, UK; RRID: SCR_012931); anti-human Osterix (anti-mouse, Cat. No. sc-393,325, RRID: AB_2895257) and anti-human Osteopontin (anti-mouse, Cat. No. sc-21,742, RRID: AB_2194997) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA; RRID: SCR_008987); secondary antibody Alexa Fluor 488-labelled (anti-rabbit, Cat. No. A21206, RRID: AB_2535792) was supplied by Molecular Probes (Invitrogen, Carlsbad, CA, USA; RRID: SCR_013318).

    Techniques: Activation Assay, Shear, Western Blot, Staining, Cell Culture

    Transplanted BM-MSCs submitted to mechanical stimulation (SS) accelerate closure of critical bone defects in an “in vivo” animal model compared to non-stimulated cells and this effect depends on tonic P2X7 and P2Y 6 receptors activation. Panel A shows a schematic representation of a rat femur with a critical bone defect drilled in the greater trochanter prepared to receive the encapsulated Pm BM-MSCs (for details see Materials and Methods). Panel B shows representative micrograph sections of critical bone defects in rat femora filled with collagen I-encapsulated BM-MSCs of a 60-years old woman. Before xenotransplantation, BM-MSCs were differentiated using an osteoblast-inducing medium for 21 days in the absence (CTR) or presence of shear stress (SS) mechanical stimulation only or together with of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Shown are images stained with hematoxylin and eosin (HE) and Masson’s trichrome highlight the bone defect area (dashed yellow line). The mesenchymal tissue (MT) consists of BM-MSCs transplanted and/or recruited to the injury site; endochondral ossification (EO) is evidenced as the result of secondary bone repair. Panel C shows three graphs computed from histological digital images, representing: (i) the percentage of MSCs area, endochondral ossification area and woven bone area as a function of the bone defect filled area (100%); (ii) the ratio between the area occupied by BM-MSCs and the total bone defect area; and (iii) the ratio between the endochondral ossification area and the total bone defect area, obtained for each experimental condition. Boxes and whiskers represent pooled data from 4 individual experiments; the cells used were obtained from 4 Pm women (60, 68, 76 and 79 years old); * P < 0.05 (ordinary one-way ANOVA with Fisher’s LSD test) represents significant differences. The insert part in panel Ci shows a higher magnification (410x) of a micrograph section stained with Masson’s trichrome in which the bone defect area shows spicules of newly formed woven bone (WB) through endochondral ossification (EO)

    Journal: Stem Cell Research & Therapy

    Article Title: Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women

    doi: 10.1186/s13287-024-03775-4

    Figure Lengend Snippet: Transplanted BM-MSCs submitted to mechanical stimulation (SS) accelerate closure of critical bone defects in an “in vivo” animal model compared to non-stimulated cells and this effect depends on tonic P2X7 and P2Y 6 receptors activation. Panel A shows a schematic representation of a rat femur with a critical bone defect drilled in the greater trochanter prepared to receive the encapsulated Pm BM-MSCs (for details see Materials and Methods). Panel B shows representative micrograph sections of critical bone defects in rat femora filled with collagen I-encapsulated BM-MSCs of a 60-years old woman. Before xenotransplantation, BM-MSCs were differentiated using an osteoblast-inducing medium for 21 days in the absence (CTR) or presence of shear stress (SS) mechanical stimulation only or together with of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Shown are images stained with hematoxylin and eosin (HE) and Masson’s trichrome highlight the bone defect area (dashed yellow line). The mesenchymal tissue (MT) consists of BM-MSCs transplanted and/or recruited to the injury site; endochondral ossification (EO) is evidenced as the result of secondary bone repair. Panel C shows three graphs computed from histological digital images, representing: (i) the percentage of MSCs area, endochondral ossification area and woven bone area as a function of the bone defect filled area (100%); (ii) the ratio between the area occupied by BM-MSCs and the total bone defect area; and (iii) the ratio between the endochondral ossification area and the total bone defect area, obtained for each experimental condition. Boxes and whiskers represent pooled data from 4 individual experiments; the cells used were obtained from 4 Pm women (60, 68, 76 and 79 years old); * P < 0.05 (ordinary one-way ANOVA with Fisher’s LSD test) represents significant differences. The insert part in panel Ci shows a higher magnification (410x) of a micrograph section stained with Masson’s trichrome in which the bone defect area shows spicules of newly formed woven bone (WB) through endochondral ossification (EO)

    Article Snippet: Anti-human P2X7 (anti-rabbit, Cat. No. APR-008, RRID: AB_2040065) and P2Y 6 (anti-rabbit, Cat. No. APR-011, RRID: AB_2040082) were purchased from Alomone (Jerusalem, Israel); anti-human Osterix (anti-rabbit, Cat No. ab22552, RRID: AB_2194492), anti-human β-Tubulin (anti-rabbit, Cat. No. ab6046, RRID: AB_2210370) and horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit, Cat. No. ab7083, RRID: AB_955416 and anti-mouse, Cat No. ab6820, RRID: AB_955438) were purchased from Abcam (Cambridge, UK; RRID: SCR_012931); anti-human Osterix (anti-mouse, Cat. No. sc-393,325, RRID: AB_2895257) and anti-human Osteopontin (anti-mouse, Cat. No. sc-21,742, RRID: AB_2194997) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA; RRID: SCR_008987); secondary antibody Alexa Fluor 488-labelled (anti-rabbit, Cat. No. A21206, RRID: AB_2535792) was supplied by Molecular Probes (Invitrogen, Carlsbad, CA, USA; RRID: SCR_013318).

    Techniques: In Vivo, Animal Model, Activation Assay, Shear, Staining

    Effect of mechanical stimulation (SS) on the density of the P2X7 and P2Y 6 receptors immunoreactivity in cultured BM-MSCs from Pm women. Panel A presents representative immunofluorescence confocal micrographs of BM-MSCs from a Pm woman (69 years old) grown for 7 and 21 days in an osteogenic-inducing medium stained against P2X7 and P2Y 6 receptors (green). Blue dots represent nuclei stained with DAPI. Scale bar is 50 μm. In panel B , ordinates represent the fluorescence intensity per cell (arbitrary units, a.u.) of the indicated immunotarget as a function of the number of days in culture (day 7 and 21). Boxes and whiskers represent pooled data from a total of 125–317 cells analysed from 4 Pm women (75 ± 2 years old). * P < 0.05 and **** P < 0.0001 (non-parametric Kruskal-Wallis test with Dunn’s multiple comparison test) represent significant differences

    Journal: Stem Cell Research & Therapy

    Article Title: Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women

    doi: 10.1186/s13287-024-03775-4

    Figure Lengend Snippet: Effect of mechanical stimulation (SS) on the density of the P2X7 and P2Y 6 receptors immunoreactivity in cultured BM-MSCs from Pm women. Panel A presents representative immunofluorescence confocal micrographs of BM-MSCs from a Pm woman (69 years old) grown for 7 and 21 days in an osteogenic-inducing medium stained against P2X7 and P2Y 6 receptors (green). Blue dots represent nuclei stained with DAPI. Scale bar is 50 μm. In panel B , ordinates represent the fluorescence intensity per cell (arbitrary units, a.u.) of the indicated immunotarget as a function of the number of days in culture (day 7 and 21). Boxes and whiskers represent pooled data from a total of 125–317 cells analysed from 4 Pm women (75 ± 2 years old). * P < 0.05 and **** P < 0.0001 (non-parametric Kruskal-Wallis test with Dunn’s multiple comparison test) represent significant differences

    Article Snippet: Anti-human P2X7 (anti-rabbit, Cat. No. APR-008, RRID: AB_2040065) and P2Y 6 (anti-rabbit, Cat. No. APR-011, RRID: AB_2040082) were purchased from Alomone (Jerusalem, Israel); anti-human Osterix (anti-rabbit, Cat No. ab22552, RRID: AB_2194492), anti-human β-Tubulin (anti-rabbit, Cat. No. ab6046, RRID: AB_2210370) and horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit, Cat. No. ab7083, RRID: AB_955416 and anti-mouse, Cat No. ab6820, RRID: AB_955438) were purchased from Abcam (Cambridge, UK; RRID: SCR_012931); anti-human Osterix (anti-mouse, Cat. No. sc-393,325, RRID: AB_2895257) and anti-human Osteopontin (anti-mouse, Cat. No. sc-21,742, RRID: AB_2194997) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA; RRID: SCR_008987); secondary antibody Alexa Fluor 488-labelled (anti-rabbit, Cat. No. A21206, RRID: AB_2535792) was supplied by Molecular Probes (Invitrogen, Carlsbad, CA, USA; RRID: SCR_013318).

    Techniques: Cell Culture, Immunofluorescence, Staining, Fluorescence, Comparison

    Mechanical stimulation facilitates the osteogenic differentiation and mineralization of cultured BM-MSCs from Pm women via the activation of P2X7 and P2Y 6 receptors. BM-MSCs grown in an osteoblast-inducing medium for 7, 14, 21 and 35 days, either in the absence or presence of shear-stress mechanical stimulation. Mechanically-stimulated Pm BM-MSCs were also tested in the absence (SS group) and in the presence of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Panel A shows the ALP activity represented in nmol/min/MTT. Zero represents identity between represented conditions and the ALP activity obtained in non-stimulated (CTR) cells (horizontal dash line); control ALP activity values were 3.5 ± 0.4, 4.3 ± 1.0, 4.6 ± 0.6 nmol/min/MTT on days 7, 14 and 21, respectively. Scatter dot plot (with mean ± S.E.M.) represent pooled data from 11 Pm women (71 ± 3 years old); seven to sixteen replicates were performed per individual. a P <0.05 (non-parametric Kruskal-Wallis test with uncorrected Dunn’s test) represent significant differences. Panel B shows the extracellular matrix mineralization at culture day 35. Zero represents identity between represented conditions and the total mineralized area values obtained in non-stimulated (CTR) cells (horizontal dash line); the total mineralized area in control cultures was 64,972 ± 14,792 µm 2 . Scatter dot plot (with mean ± S.E.M.) represent pooled data from 8 Pm women (70 ± 3 years old); four to eight replicates were performed per individual. b P <0.01, c P <0.001 and d P <0.0001 (non-parametric Kruskal-Wallis test with Dunn’s multiple comparison test) represent significant differences. Right hand-side images show typical micrographs displaying bone nodule formation (red-brownish spots) in BM-MSC cultures from a Pm woman submitted (SS) or not (CTR) to mechanical stimulation either in the absence or presence of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Scale bar is 50 μm

    Journal: Stem Cell Research & Therapy

    Article Title: Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women

    doi: 10.1186/s13287-024-03775-4

    Figure Lengend Snippet: Mechanical stimulation facilitates the osteogenic differentiation and mineralization of cultured BM-MSCs from Pm women via the activation of P2X7 and P2Y 6 receptors. BM-MSCs grown in an osteoblast-inducing medium for 7, 14, 21 and 35 days, either in the absence or presence of shear-stress mechanical stimulation. Mechanically-stimulated Pm BM-MSCs were also tested in the absence (SS group) and in the presence of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Panel A shows the ALP activity represented in nmol/min/MTT. Zero represents identity between represented conditions and the ALP activity obtained in non-stimulated (CTR) cells (horizontal dash line); control ALP activity values were 3.5 ± 0.4, 4.3 ± 1.0, 4.6 ± 0.6 nmol/min/MTT on days 7, 14 and 21, respectively. Scatter dot plot (with mean ± S.E.M.) represent pooled data from 11 Pm women (71 ± 3 years old); seven to sixteen replicates were performed per individual. a P <0.05 (non-parametric Kruskal-Wallis test with uncorrected Dunn’s test) represent significant differences. Panel B shows the extracellular matrix mineralization at culture day 35. Zero represents identity between represented conditions and the total mineralized area values obtained in non-stimulated (CTR) cells (horizontal dash line); the total mineralized area in control cultures was 64,972 ± 14,792 µm 2 . Scatter dot plot (with mean ± S.E.M.) represent pooled data from 8 Pm women (70 ± 3 years old); four to eight replicates were performed per individual. b P <0.01, c P <0.001 and d P <0.0001 (non-parametric Kruskal-Wallis test with Dunn’s multiple comparison test) represent significant differences. Right hand-side images show typical micrographs displaying bone nodule formation (red-brownish spots) in BM-MSC cultures from a Pm woman submitted (SS) or not (CTR) to mechanical stimulation either in the absence or presence of selective P2X7 (SS + A438079, 3 µM) and P2Y 6 (SS + MRS 2578, 0.1 µM) receptor antagonists. Scale bar is 50 μm

    Article Snippet: Anti-human P2X7 (anti-rabbit, Cat. No. APR-008, RRID: AB_2040065) and P2Y 6 (anti-rabbit, Cat. No. APR-011, RRID: AB_2040082) were purchased from Alomone (Jerusalem, Israel); anti-human Osterix (anti-rabbit, Cat No. ab22552, RRID: AB_2194492), anti-human β-Tubulin (anti-rabbit, Cat. No. ab6046, RRID: AB_2210370) and horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit, Cat. No. ab7083, RRID: AB_955416 and anti-mouse, Cat No. ab6820, RRID: AB_955438) were purchased from Abcam (Cambridge, UK; RRID: SCR_012931); anti-human Osterix (anti-mouse, Cat. No. sc-393,325, RRID: AB_2895257) and anti-human Osteopontin (anti-mouse, Cat. No. sc-21,742, RRID: AB_2194997) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA; RRID: SCR_008987); secondary antibody Alexa Fluor 488-labelled (anti-rabbit, Cat. No. A21206, RRID: AB_2535792) was supplied by Molecular Probes (Invitrogen, Carlsbad, CA, USA; RRID: SCR_013318).

    Techniques: Cell Culture, Activation Assay, Shear, Activity Assay, Control, Comparison

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    Confocal microscope images demonstrating expression of P2Y 6 (FITC, green) in cross‐sections of middle cerebral arteries. DAPI (4′,6‐Diamidine‐2′‐phenylindole dihydrochloride, a blue nucleus stain, has been added. The tMCAO, Sham, and SAH images were generated by superimposing a DAPI stained image on a FITC stained image. The negative control demonstrates the lack of unspecific antibody binding and the autofluorescence of the lamina elastica interna which separates the intima from the underlying layer of smooth muscle cells

    Journal: Physiological Reports

    Article Title: Changes in P2Y 6 receptor‐mediated vasoreactivity following focal and global ischemia

    doi: 10.14814/phy2.15283

    Figure Lengend Snippet: Confocal microscope images demonstrating expression of P2Y 6 (FITC, green) in cross‐sections of middle cerebral arteries. DAPI (4′,6‐Diamidine‐2′‐phenylindole dihydrochloride, a blue nucleus stain, has been added. The tMCAO, Sham, and SAH images were generated by superimposing a DAPI stained image on a FITC stained image. The negative control demonstrates the lack of unspecific antibody binding and the autofluorescence of the lamina elastica interna which separates the intima from the underlying layer of smooth muscle cells

    Article Snippet: The cell suspensions were stained overnight at +4°C temperature with primary goat anti‐SM22α (1:100, Abcam) and primary rabbit anti‐P2Y 6 (1:100, Alomone), respectively or goat isotope control IgG (Abcam), rabbit isotope control IgG (Abcam) in blocking buffer (PBS + 5% normal donkey serum) protected from light with rocking, respectively.

    Techniques: Microscopy, Expressing, Staining, Generated, Negative Control, Binding Assay

    Bar graph with scatter plot demonstrating the expression of P2Y 6 receptors on vascular smooth muscle cells determined by flow cytometry. Data are presented as median and IQR, n = 3–6, * p < 0.05

    Journal: Physiological Reports

    Article Title: Changes in P2Y 6 receptor‐mediated vasoreactivity following focal and global ischemia

    doi: 10.14814/phy2.15283

    Figure Lengend Snippet: Bar graph with scatter plot demonstrating the expression of P2Y 6 receptors on vascular smooth muscle cells determined by flow cytometry. Data are presented as median and IQR, n = 3–6, * p < 0.05

    Article Snippet: The cell suspensions were stained overnight at +4°C temperature with primary goat anti‐SM22α (1:100, Abcam) and primary rabbit anti‐P2Y 6 (1:100, Alomone), respectively or goat isotope control IgG (Abcam), rabbit isotope control IgG (Abcam) in blocking buffer (PBS + 5% normal donkey serum) protected from light with rocking, respectively.

    Techniques: Expressing, Flow Cytometry

    Representative dot plots and histograms of the experimental setup and analysis of flow cytometry data. (a) Dot plot histogram for entire cell population after isolation. (b) Dot plot histogram of single cells population according to FSC‐H (H—high) versus FSC‐A (A—area scaling). (c) Representative histogram of negative single cells population for Fixable Viability Dye eFluor 780 (viable cells, live/dead); (d) Representative histogram demonstrating SM22α‐positive events of viable cells suspension (log scale). (e) Representative histograms demonstrating P2Y 6 ‐positive events of viable VSMC suspension (log scale)

    Journal: Physiological Reports

    Article Title: Changes in P2Y 6 receptor‐mediated vasoreactivity following focal and global ischemia

    doi: 10.14814/phy2.15283

    Figure Lengend Snippet: Representative dot plots and histograms of the experimental setup and analysis of flow cytometry data. (a) Dot plot histogram for entire cell population after isolation. (b) Dot plot histogram of single cells population according to FSC‐H (H—high) versus FSC‐A (A—area scaling). (c) Representative histogram of negative single cells population for Fixable Viability Dye eFluor 780 (viable cells, live/dead); (d) Representative histogram demonstrating SM22α‐positive events of viable cells suspension (log scale). (e) Representative histograms demonstrating P2Y 6 ‐positive events of viable VSMC suspension (log scale)

    Article Snippet: The cell suspensions were stained overnight at +4°C temperature with primary goat anti‐SM22α (1:100, Abcam) and primary rabbit anti‐P2Y 6 (1:100, Alomone), respectively or goat isotope control IgG (Abcam), rabbit isotope control IgG (Abcam) in blocking buffer (PBS + 5% normal donkey serum) protected from light with rocking, respectively.

    Techniques: Flow Cytometry, Isolation