anti p2y 1 r antibody  (Alomone Labs)

Journal: Purinergic Signalling

Article Title: Machine learning-aided search for ligands of P2Y 6 and other P2Y receptors

doi: 10.1007/s11302-024-10003-4

Figure Lengend Snippet: Comparison of functional effects (in a FLIPR assay of calcium transients) at the hP2Y 1 R stably expressed in 1321N1 astrocytoma cells. A, B Effects of ABBV-744 and several other ML-selected compounds on concentration-dependent hP2Y 1 R activation by selective nucleotide agonist MRS2365: A 30 µM ABBV-744, AZD3695, and AZD5423; B 100 µM ABBV-744, AZD3695, and AZD5423. C Effects of ABBV-744 and ABBV-075 on the activation of the hP2Y 1 R

Article Snippet: Selective P2Y 1 R agonist MRS2365 ([[(1 R ,2 R ,3 S ,4 R ,5 S )-4-[6-amino-2-(methylthio)-9 H -purin-9-yl]-2,3-dihydroxybicyclo-[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt) was from Tocris (Minneapolis, MN).

Techniques: Comparison, Functional Assay, Stable Transfection, Concentration Assay, Activation Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Changes of C3d/S100A10 levels, and pan-reactive and A1/A2 specific gene expression in reactive astrocytes after treatment with microglia-conditioned media (MCM) and P2Y 1 R-ANT. (a), (b). Representative images (a) and quantitative data (b) of western blots showing protein levels of GFAP, C3d, S100A10, and CSPG in non-OGD astrocytes, OGD astrocytes, OGD astrocytes treated with MCM, OGD astrocytes treated with P2Y 1 R-ANT, and OGD astrocytes treated with P2Y 1 R-ANT and MCM. β-actin was used as an internal control. * P < 0.05 vs. non-OGD astrocyte, # P < 0.05 vs. OGD astrocytes. (c). Venn diagram of mRNAs with upregulated (fold change ≥ 1.5) and downregulated (fold change ≤ 0.67) expressions in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (d). Heatmap of the entire mRNA expression in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (e). Heatmaps comparing the mean expression of pan-reactive, A1-specific, and A2 specific genes in OGD astrocytes; OGD astrocytes treated with P2Y 1 R-ANT (1 mM); and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. N = 4/group. Values are the mean ± SD. GFAP = glial fibrillary acidic protein, CSPG = chondroitin sulfate proteoglycans, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Expressing, Western Blot, Control

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Change in inflammatory gene expression and pathway analysis. (a). Top 20 significant canonical pathways of the core analysis in IPA of most highly expressed genes in OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM relative to OGD astrocytes. Blue bars: negative z-score; orange bars: positive z-score; gray bars: no activity pattern available; white bars: activity of zero. (b), (c). Heatmap of mRNA-related Neuroinflammation Signaling expression, and quantitative analysis of representative mRNA-related Neuroinflammation Signaling in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. * P < 0.05 vs. OGD astrocyte, # P < 0.05 vs. OGD astrocytes treated with P2Y 1 R-ANT. (d). Signaling pathway predicted by analyzing changes using IPA software in mRNA expression in the OGD astrocytes treated with P2Y 1 R-ANT and MCM relative to OGD astrocytes, or OGD astrocytes treated with P2Y 1 R-ANT. The functional networks were generated via IPA (QIAGEN Inc., https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis ). N = 4/group. Values are the mean ± SD. IPA = Ingenuity Pathway Analysis, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium, MAPK = mitogen-activated protein kinase, NF-κB = nuclear factor-κβ, TNF-α = tumor necrosis factor, IL-1β = interleukin-1β, NOX = nitrogen oxides, CASP8 = Caspase 8

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Expressing, Activity Assay, Software, Functional Assay, Generated

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Significance of AEVs derived from anti-inflammatory astrocytes to the peri-infarct area after MCAO. (a). The experimental scheme of isolating AEVs from OGD astrocytes treated with MCM and P2Y 1 R-ANT and their application for rats subjected to MCAO and cultured cortical neurons. (b), (c). Modified neurological severity score ( b ) and latency to fall off the rotarod at 56 days after MCAO ( c ) in PBS treatment, treatment with AEVs derived from OGD astrocytes (100 µg), and OGD astrocytes treated with MCM and P2Y 1 R-ANT (100 µg) in rats subjected to MCAO. N = 6–7/group. Values are the mean ± SD. (d), (e). Double immunofluorescent confocal images and quantitative data of the peri-infarct area at 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing C3d + area (green) ( d ) and S100A10 + area (green) ( e ), and co-localized with GFAP + area (yellow). Merge ratio of C3d /GFAP ( d ), and S100A10/GFAP ( e ). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats, # P < 0.05 vs. rats treated with AEVs derived from OGD astrocytes. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, OGD = oxygen–glucose deprivation

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Derivative Assay, Cell Culture, Modification

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Expression of microRNAs (miRNAs) in AEVs and inflammatory regulation in peri-infarct glial scars. (a). Heatmap of miRNA profiles on AEVs derived from OGD astrocytes and OGD astrocytes treated with P2Y 1 R-ANT and MCM. (b). Quantitative analysis of representative miRNAs related to ‘Inflammatory Response’ in AEVs derived from OGD astrocytes treated with P2Y 1 R-ANT and MCM, relative to AEVs derived from OGD astrocytes. N = 4/group. Values are the mean ± SD. * P < 0.05 vs. AEVs derived from OGD astrocytes. (c), (d). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing TNFα + area (green) ( c ) and NF-κB + area (green) ( d ), and co-localized with GFAP + area (yellow). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, P2Y 1 R-ANT = P2Y 1 receptor antagonist

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Expressing, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Axonal outgrowth after AEV treatment and hindering by TNF-α (a), (b). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing pNFH + axons (green) ( a ) and MAP2 cells + (green) ( b ), with GFAP. N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. Scale bar = 100 μm. (c). Representative time-lapse microscopic images and quantitative data of primary cortical neurons in a microfluidic chamber showing axonal elongation (distance from yellow arrow to red arrow) in OGD neurons, OGD neurons treated with 1 ng/µl of TNF-α, and OGD neurons treated with 10 ng/µl of TNF-α. N = 3/group. Values are the mean ± SD. Scale bar = 20 μm. Quantitative data of axonal elongation per 30 min prior to 96 h after OGD. * P < 0.05, OGD neurons treated with 1 ng/µL of TNF-α vs. OGD neurons; # P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons; $ P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons treated with 1 ng/µL of TNF-α. AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, pNFH = phosphorylated neurofilament heavy chain

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Diagram depicting the findings of the present study. Microglia and inhibition of P2Y 1 R regulate reactive astrocytes by transforming C3d/S100A10 expression and suppressing neuroinflammation. AEVs derived from reactive astrocytes with anti-inflammatory properties possessing miR-146a-5p regulate glial scars by suppressing NF-κB and TNF-α, which is permissive for axonal outgrowth and improves stroke recovery

Article Snippet: The primary antibodies used in this study were goat polyclonal anti-GFAP (1:40,000; Abcam), goat polyclonal anti-C3d (a marker for A1 astrocytes) (1:2,000, R&D Systems), rabbit polyclonal anti-S100A10 (1:2,000, Proteintech), mouse monoclonal anti-CSPG (1:100,000, Abcam), mouse monoclonal anti-phosphorylated neurofilament heavy chain (pNFH) (1:500; BioLegend), rabbit polyclonal anti-P2Y 1 R-ANT (1:200; Alomone Labs), rabbit polyclonal anti-caspase-3 (1:500, Abcam), and rabbit monoclonal anti-actin (1:10,000, Abcam).

Techniques: Inhibition, Expressing, Derivative Assay