Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) RNAscope in-situ hybridization of P2Y 2 mRNA expression (magenta) in tumor and matching normal adjacent tissue. ( B ) P2Y 2 mRNA expression in tumor (TCGA) and normal (GTEx) pancreatic tissue samples (*p<0.0001). Graph generated using GEPIA. ( C ) Kaplan-Meier plot comparing patients with high vs low expression of P2Y 2 in the Pancreatic Adenocarcinoma (PAAD) The Cancer Genome Atlas (TCGA) cohort. Graph generated using KMplot. ( D ) Top result of a GSEA (performed with WebGestalt) of two different pancreatic adenocarcinoma patient cohorts (PAAD TCGA and PDAC CPTAC) for the PANTHER pathway functional database. ( E ) Incucyte images of the pancreatic cancer cell line AsPC-1 12 hr after treatment with 100 µM ATP alone or with 5 µM AR-C (P2Y 2 antagonist). Cells are transduced with Lifeact to visualize f-actin (green). ( F ) Schematic of the amino acid sequence of P2Y 2 showing an RGD motif in the first extracellular loop (image generated in http://gpcrdb.org/ ). ( G ) IF staining of P2Y 2 (green), integrin αV (red), and DAPI (blue) in AsPC-1 cells showing colocalization of P2Y 2 and integrin αV (yellow).

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: In Situ Hybridization, Expressing, Generated, Functional Assay, Transduction, Sequencing, Staining

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) RNAscope in-situ hybridization of a positive control ( PPIB , Cyclophilin B), negative control ( DapB ), and P2Y 2 mRNA expression in a PDAC tissue slide showing tumor and normal adjacent tissue. ( B ) Single-cell expression of P2Y 2 in healthy pancreatic tissue from the Human Protein Atlas ( https://www.proteinatlas.org/ENSG00000175591-P2RY2/single+cell+type/pancreas ). ( C ) Top four results of a GSEA (performed with WebGestalt) of two different pancreatic adenocarcinoma patient cohorts (PAAD TCGA and PDAC CPTAC) for the ‘Molecular Function’ Gene Ontology (GO) functional database. ( D ) Incucyte analysis of average object area related to the average cell area of AsPC-1 cells at different concentrations of ATP (error bars show standard deviation). ( E ) Incucyte images of AsPC-1 cells with different concentrations of AR-C with or without ATP. ( F ) IF staining of four different PDAC cell lines showing various levels of P2Y 2 (green) and integrin αV (red) protein expression. ( G ) The respective reads per kilobase of exon per million reads mapped (RPKM) from CCLE.

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: In Situ Hybridization, Positive Control, Negative Control, Expressing, Functional Assay, Standard Deviation, Staining

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) Schematic diagram of the hanging drop sphere model for 3D sphere invasion assays. ( B ) Bright field and fluorescent images of spheres formed using AsPC-1 cells (magenta) with a histone 2B (H2B) tagged with a red fluorescent protein (RFP) and the stellate cell line PS-1 (green) with H2B tagged with a green fluorescent protein (GFP). Middle panel shows AsPC-1 cells in spheres with a dotted line highlighting the central sphere area. Spheres were treated with vehicle control or 100 µM ATP alone or with 5 µM AR-C or 10 µM cRGDfV. The quantification is shown in ( C ) using SuperPlots, where each color represents a biological repeat (n=3) and the larger points represent the mean % Invasion for each repeat. ( D ) Quantification of spheres formed by AsPC-1 cells transfected with a control siRNA or P2Y 2 siRNA and treated with or without 100 µM ATP. ( E ) Bright field and fluorescent images of spheres formed by AsPC-1 cells subjected to CRISPR/Cas9 gene disruption using a control guide RNA (CTR CRISPR ) or P2Y 2 guide RNAs (P2Y 2 CRISPR ) and treated with or without 100 µM ATP. Quantification in ( F ). ( G, I ) Bright field and fluorescent images of AsPC-1 P2Y 2 CRISPR cells or PANC-1 cells (respectively) transfected with wild-type P2RY2 (P2Y 2 RGD ) or mutant P2RY2 D97E (P2Y 2 RGE ) treated with or without 100 µM ATP and its quantification in ( H ) and ( J ), respectively. Statistical analysis with Kuskal-Wallis multiple comparison tests.

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: Transfection, CRISPR, Mutagenesis

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A, B ) Overview of the DNA-PAINT microscopy technique and qPAINT analysis pipeline. ( C ) Histogram of qPAINT indices for αV (blue) and P2Y 2 (red) single-molecule localization clusters. Solid lines represent multi-peak Gaussian fit. ( D ) Rendered DNA-PAINT images of AsPC-1 P2Y 2 CRISPR cells transfected with P2Y 2 RGD or P2Y 2 RGE with or without 100 µM of ATP and close-ups showing the protein maps reconstructed from DNA-PAINT localization maps of P2Y 2 (red) and integrin αV (cyan). The quantification of the number of proteins or protein clusters (>3 proteins) in each region of interest (ROI) are for P2Y 2 (red) ( E ) and ( F ), respectively and integrin αV (cyan) ( G ) and ( H ), respectively. Quantification of protein proximity using the nearest neighbor distance (NND), with the percentages of integrin αV and P2Y 2 proteins being <50 nm apart ( I ), between different αV integrins being 20–100 nm ( J ) or <20 nm ( K ) apart; and P2Y 2 from other P2Y 2 proteins being <40 nm apart ( H ). Statistical analysis with Kuskal-Wallis multiple comparison test of 21 4x4 µm ROIs from a minimum of 5 cell regions per condition.

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: Microscopy, CRISPR, Transfection

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) Schematic diagram of the predicted maximum distance between fluorescent molecules indicating physical contact between proteins, to the nearest first significant figure. ( B ) Histograms of the nearest neighbor distance between proteins vs the frequency of occurrence for AsPC-1 P2Y 2 CRISPR in different conditions (solid line, strong color) or randomly computer-generated controls (dotted line, light color).

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: CRISPR, Generated

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: Proposed mechanism of P2Y 2 and integrin interactions in pancreatic cancer invasion.

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques:

Journal: PLoS ONE

Article Title: Microglial P2Y 12 Deficiency/Inhibition Protects against Brain Ischemia

doi: 10.1371/journal.pone.0070927

Figure Lengend Snippet: P2Y 12 was knocked down in BV2 cells using siRNA, and were cocultured with mixed neuron-astrocyte cultures. Scrambled siRNA was used as a control. Following OGD, neuron viability was decreased in BV2 cells receiving control siRNA, but was increased where P2Y 12 was deficient (A). The tendency towards microglial clustering was increased by OGD, but prevented by P2Y 12 deficiency (B, C). Immunostains of the microglial marker isolectin B4 (IB4) indicate that the majority of the cells forming the clusters are indeed microglia (C). **P<0.01.

Article Snippet: The membranes were probed with an antibody against P2Y 12 (1∶250 dilution, #APR-012, Alomond Labs), followed by secondary antibody then reacted with ECL.

Techniques: Marker

Journal: PLoS ONE

Article Title: Microglial P2Y 12 Deficiency/Inhibition Protects against Brain Ischemia

doi: 10.1371/journal.pone.0070927

Figure Lengend Snippet: A: A Boyden-like assay was used to estimate the ability of BV2 cells to travel through a cell culture insert. BV2 cells were placed in inserts, which were then placed above wells containing various potential chemotactic stimulants. Cells migrating into the lower chamber were then quantified using a fluorescent detection dye. Test media are indicated in the upper row of the x-axis label (media = BSS 5.5; AST = astrocyte-conditioned media; Neu = neuron-conditioned media). Cultures previously exposed to OGD are indicated by ‘+’, and those not exposed by ‘−’. OGD increased BV2 cell migration neuron conditioned media, but not astrocyte conditioned media. B: Using a Dunn chamber assay where distance traveled can be estimated, primary microglia harvested from wildtype (WT) or P2Y 12 deficient (P2Y 12 −/− , KO) mice, and were exposed to conditioned media from OGD-exposed wildtype neurons. The distance traveled over a 30 minute observation period is shown. Primary microglia from KO mice traveled shorter distances than WT microglia. (*P<0.05, **P<0.01).

Article Snippet: The membranes were probed with an antibody against P2Y 12 (1∶250 dilution, #APR-012, Alomond Labs), followed by secondary antibody then reacted with ECL.

Techniques: Cell Culture, Migration, Boyden Chamber Assay

Journal: PLoS ONE

Article Title: Microglial P2Y 12 Deficiency/Inhibition Protects against Brain Ischemia

doi: 10.1371/journal.pone.0070927

Figure Lengend Snippet: Mortality & PComm scores in P2Y 12 deficient & Wildtype mice.

Article Snippet: The membranes were probed with an antibody against P2Y 12 (1∶250 dilution, #APR-012, Alomond Labs), followed by secondary antibody then reacted with ECL.

Techniques:

Journal: PLoS ONE

Article Title: Microglial P2Y 12 Deficiency/Inhibition Protects against Brain Ischemia

doi: 10.1371/journal.pone.0070927

Figure Lengend Snippet: CBF during BCCAO by LDF.

Article Snippet: The membranes were probed with an antibody against P2Y 12 (1∶250 dilution, #APR-012, Alomond Labs), followed by secondary antibody then reacted with ECL.

Techniques:

Journal: PLoS ONE

Article Title: Microglial P2Y 12 Deficiency/Inhibition Protects against Brain Ischemia

doi: 10.1371/journal.pone.0070927

Figure Lengend Snippet: Following bilateral common carotid artery occlusion for 12 min followed by 3 d reperfusion, P2Y 12 deficient mice (P2Y 12 +/− ) suffered less injury to hippocampal CA1 compared to wildtype (WT) mice. WT mice treated with clopidogrel (WT+CL, Clopidogrel) were also protected (A, B). Following BCCAO, microglial densities as estimated from counts of isolectin B4 (IB4) positive cells were also decreased within CA1 among P2Y 12 +/− and Clopidogrel treated mice (A, C). *P<0.05, **P<0.01.

Article Snippet: The membranes were probed with an antibody against P2Y 12 (1∶250 dilution, #APR-012, Alomond Labs), followed by secondary antibody then reacted with ECL.

Techniques:

Journal: PLoS ONE

Article Title: Microglial P2Y 12 Deficiency/Inhibition Protects against Brain Ischemia

doi: 10.1371/journal.pone.0070927

Figure Lengend Snippet: Numbers of cells positive for NF κ B’s p65 subunit were decreased among P2Y 12 +/− and treated mice (A, C). Furthermore, cells of P2Y 12 +/− and treated mice had less nuclear NF κ B staining (A, green arrows) compared to untreated WT following BCCAO (A, D). B: Double labeling for NF κ B’s p65 subunit (red) and the microglial marker CD11b (green) show that most CD11b cells are also positive for NF κ B after BCCAO in WT brains. Several microglia in brains of P2Y 12 +/− mice or WT mice treated with clopidogrel (WT+CL) are not NF κ B positive (arrows). *P<0.05, **P<0.01, scale bar = 25 µm.

Article Snippet: The membranes were probed with an antibody against P2Y 12 (1∶250 dilution, #APR-012, Alomond Labs), followed by secondary antibody then reacted with ECL.

Techniques: Staining, Labeling, Marker

Journal: Cell Communication and Signaling : CCS

Article Title: Bradykinin-induced Ca 2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y 12 receptors activation

doi: 10.1186/1478-811X-11-70

Figure Lengend Snippet: Bradykinin-induced [Ca 2+ ] i mobilization is partially dependent on the activation of P2Y 12 purinoceptors. Panel A shows the effect of bradykinin (BK, 30 μM) after pretreating human subcutaneous fibroblasts with apyrase (2 U/mL, Ai ), which catabolizes ATP/ADP into AMP, and after inhibition of ectonucleotidases, with POM-1 (20 μM, Aii ) or after removing Mg 2+ from the incubation fluid (Aiii) . Panel C shows the effects of BK (30 μM) in the absence or presence of selective P2Y 1 , P2Y 12 and P2Y 13 receptor antagonists, respectively MRS 2179 (0.3 μM, Ci ), AR-C 66096 (0.1 μM, Cii ) and MRS 2211 (10 μM, Ciii ). Cells were pre-incubated with the cell-permeant fluorescent calcium indicator, Fluo-4 NW (2.5 μM, see Methods). [Ca 2+ ] i transients were calibrated to the maximal calcium load produced by ionomycin (5 μM, 100% response). Black arrows indicate the time of drugs application. No changes in baseline fluorescence were observed after application of the modulators. Each point represents pooled data from an n number of experiments. The vertical bars represent S.E.M.. * p < 0.05 represent significant differences from BK (30 μM) alone. Panel B illustrates the time course of the extracellular catabolism of adenine nucleotides in human subcutaneous fibroblasts grown in culture for 11 days. ATP, ADP or AMP (3 μM) were added to the culture medium at time zero; samples (75 μl) were collected at indicated times. Each sample was analyzed by HPLC to separate and quantify ATP (white), ADP (black), AMP (grey), adenosine (ADO, red), inosine (INO, orange) and hypoxanthine (HX, green). Each point represents pooled data from two individuals; 2 replicas were performed for each individual. The calculated half-life time (t ½ ) for each initial substrate is shown for comparison. Panel D shows immunoreactivity of human subcutaneous fibroblasts against the P2Y 12 receptor; shown image is representative of three independent experiments. Image scale bar is 30 μm.

Article Snippet: Cultured cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 minutes, washed 3 times in PBS (10 minutes each) and, subsequently, incubated with blocking buffer I (10% FBS, 1% bovine serum albumin (BSA), 0.1% Triton-X, 0.05% NaN 3 ) for 1 h. Primary antibodies, diluted in blocking buffer II (5% FBS, 1% BSA, 0.1% Triton-X, 0.05% NaN 3 ), were applied [mouse anti-porcine vimentin 1:75 (DAKO); rabbit anti-human collagen I 1:50 (AbDSerotec); mouse anti-human α-smooth muscle actin-FITC 1:250 (Sigma-Aldrich); rabbit anti-human P2Y 12 1:100 (Alomone); rabbit anti-human Cx43 1:600 and rabbit anti-human Panx1 1:1000 (Abcam)] and the slides incubated overnight at 4°C.

Techniques: Activation Assay, Inhibition, Incubation, Produced, Fluorescence

Journal: PLoS ONE

Article Title: Enhancement of Glucose Uptake in Mouse Skeletal Muscle Cells and Adipocytes by P2Y 6 Receptor Agonists

doi: 10.1371/journal.pone.0116203

Figure Lengend Snippet: Structures of P2Y 6 R ligands: nucleotide agonists.

Article Snippet: P2Y 6 R specific antibody was from Alomone Labs Ltd. (Jerusalem, Israel).

Techniques:

Journal: PLoS ONE

Article Title: Enhancement of Glucose Uptake in Mouse Skeletal Muscle Cells and Adipocytes by P2Y 6 Receptor Agonists

doi: 10.1371/journal.pone.0116203

Figure Lengend Snippet: MRS2957 was applied in the presence or absence of P2Y 6 R antagonist MRS2578 (1 µM). * P <0.05, when compared to controls (n = 3).

Article Snippet: P2Y 6 R specific antibody was from Alomone Labs Ltd. (Jerusalem, Israel).

Techniques:

Journal: PLoS ONE

Article Title: Enhancement of Glucose Uptake in Mouse Skeletal Muscle Cells and Adipocytes by P2Y 6 Receptor Agonists

doi: 10.1371/journal.pone.0116203

Figure Lengend Snippet: C2C12 cells (A) or 3T3-L1 cells (B) were preincubated with inhibitors of MAPK (U0126, 10 µM) and AMPK (Compound C, 10 µM) followed by treatment with P2Y 6 R agonist MRS2957. * P <0.05, when compared to controls, # P <0.05, when compared to MRS2957, 100 nM (n = 3).

Article Snippet: P2Y 6 R specific antibody was from Alomone Labs Ltd. (Jerusalem, Israel).

Techniques:

Journal: PLoS ONE

Article Title: Differential Inhibitory Effects of CysLT 1 Receptor Antagonists on P2Y 6 Receptor-Mediated Signaling and Ion Transport in Human Bronchial Epithelia

doi: 10.1371/journal.pone.0022363

Figure Lengend Snippet: The expression of CysLT 1 (44 kDa) and P2Y 6 (41 kDa) receptors was demonstrated (lane 1), and their positions on the blot closely matched their calculated molecular masses of 39 kDa and 36 kDa, respectively. Detection of these protein bands appeared specific, as they were mostly blocked by prior reabsorption of the antibodies with their respective control antigen for 2 h at 4°C (lane 2).

Article Snippet: Membranes were blocked for 1 h at room temperature by using 1% bovine serum albumin in phosphate buffered saline containing 0.05% Tween 20 and incubated overnight at 4°C with specific CysLT 1 receptor polyclonal antibody (1∶300, rabbit polyclonal anti-CysLT 1 , no. 120500, Cayman Chemical, Ann Arbor, MI) or P2Y 6 receptor antibody (1∶200, no. APR-011, rabbit polyclonal anti-P2Y 6 , Alomone, Jerusalem, Israel).

Techniques: Expressing

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Primary antibodies employed in the study.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques:

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 antibodies validation and RT-PCR analysis. (a) Scheme of human P2RY 12 gene location, transcript variants [ , ], and protein structure, with amino acid epitopes recognized by the used antibodies and highlighted in color ( intra1 , intra2 , and intra fl , red circle; c-ter , green oval). Species conservation for each epitope was calculated by using BLAT tool of UCSC genome browser . (b) Total protein extracts from SH-SY5Y or HEK293 cells expressing Myc-tagged P2Y 12 receptor were subjected to Western blot analysis with the indicated antibodies. (c) Total protein extracted from human, rat and mouse brain, from primary mouse microglia (mMG) and rat oligodendrocyte (OL) cultures were subjected to Western blot analysis with the indicated antibodies. For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (d) RT-PCR using primers specific for P2Y 12 mRNA was performed on total RNA from rat microglia (rMG) and OL. Control lanes show RT-PCR performed without reverse transcriptase enzyme.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

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

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in dissociated and organotypic primary cultures. (a) Mouse primary cortical microglia were subjected to immunofluorescence and confocal analysis with phalloidin (green, merged field) and P2Y 12 receptor antibodies (red, insets and merged) and Hoechst (white, merged). Scale bars in insets: 20 μ m. (b) Double immunofluorescence and confocal analysis of primary rat mature (OL) and precursor (OPC) oligodendrocytes was performed with antibodies for P2Y 12 receptor, MBP, NG2 . For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (c) Rat cerebellar organotypic cultures were analyzed by double immunofluorescence and confocal microscopy for intra1 (red) and c-ter (red), highlighting different structures (see also insets), and MBP (green).

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in rat brain tissue. Double immunofluorescence and confocal analysis was performed on sections from rat cerebellum (panels (a), (b), (c), (g), (h), and (i)) and striatum (panels (d), (e), (f), (j), (k), and (l)) with intra1 , intra2 -lots AN01/02/04, intra fl , c-ter (all red), and GFAP (green, inset b1), CD11b (green, insets c1, f1, h1; yellow merged, inset i1; green, panel (k); merged, panel (l)), MBP (yellow merged, inset c2; green, inset e1; green, panels (h) and (i); green, inset j1) antibodies.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Temporal and regional pattern of P2Y 12 expression in SOD1-G93A ALS spinal microglia. (a) Double immunofluorescence and confocal analysis on lumbar spinal cord sections (L3–L5) of wild-type (WT) mice was performed with c-ter antibody (green and yellow, merged and insets), CD11b (left panel, yellow, merged and inset), and CD68 (right panel, red, merged and inset), in both dorsal (DH) and ventral (VH) horns of spinal cord. (b) Double immunofluorescence and confocal analysis on SOD1-G93A lumbar spinal cord sections (L3–L5) at two different stages of ALS disease, that is, 20 weeks, and end stage, was performed with c-ter (green) and CD68 (red) antibodies. (c) Equal amount of total lumbar spinal cord lysates (L3–L5) from WT and SOD1-G93A ( n = 4 for each group) were subjected to Western blotting and immunoreactions with c-ter and CD68 antibodies; anti- β -actin was used for protein normalization. Data represent means ± SEM. Statistical significance was calculated by Student's t -test, * P < 0.05.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in human cortex. Sections from human healthy and SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for the immunoreactive markers c-ter (red, panels (a), (c); insets a1, a2, c1; yellow merged, inset c2), intra1 (red, panels (d), insets d1, d2, d3; yellow merged, panel f, insets f1, f2), MBP (green, panels (b), (c), (e), insets c1, e1; yellow merged, panel (f), inset f1), MHC II (green, inset d3), and integrin α II/ β 3 (green, insets b2, e2; yellow merged, insets c2, f2). The asterisks show decreased P2Y 12 immunoreactivity in proximity to MS lesion.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Regional distribution of P2Y 12 in proximity to MS lesions. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for c-ter (red) and MHC II (green) immunoreactivity. In proximity to the demyelinating active cortical lesion expressing augmented positivity for MHC II, microglia gradually lose immunoreactivity for c-ter antibody. Microglia express differential immunoreactivity in the four chosen areas which are found inside (circled b-c) and around (circled a–d) a lesion.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Draw of microglial marker expression as a function of activation. Branched microglia are represented in blue and activated microglia in red. Iba1 , CD11b, and MHC II are mostly expressed in microglia throughout the different morphological states and their expression increases during activation (light blue to red color). P2Y 12 c-ter (light blue) and CD68 (red) are expressed, respectively, in branched or roundish/activated microglia.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Marker, Expressing, Activation Assay

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Primary antibodies employed in the study.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques:

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 antibodies validation and RT-PCR analysis. (a) Scheme of human P2RY 12 gene location, transcript variants [ , ], and protein structure, with amino acid epitopes recognized by the used antibodies and highlighted in color ( intra1 , intra2 , and intra fl , red circle; c-ter , green oval). Species conservation for each epitope was calculated by using BLAT tool of UCSC genome browser . (b) Total protein extracts from SH-SY5Y or HEK293 cells expressing Myc-tagged P2Y 12 receptor were subjected to Western blot analysis with the indicated antibodies. (c) Total protein extracted from human, rat and mouse brain, from primary mouse microglia (mMG) and rat oligodendrocyte (OL) cultures were subjected to Western blot analysis with the indicated antibodies. For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (d) RT-PCR using primers specific for P2Y 12 mRNA was performed on total RNA from rat microglia (rMG) and OL. Control lanes show RT-PCR performed without reverse transcriptase enzyme.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

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

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in dissociated and organotypic primary cultures. (a) Mouse primary cortical microglia were subjected to immunofluorescence and confocal analysis with phalloidin (green, merged field) and P2Y 12 receptor antibodies (red, insets and merged) and Hoechst (white, merged). Scale bars in insets: 20 μ m. (b) Double immunofluorescence and confocal analysis of primary rat mature (OL) and precursor (OPC) oligodendrocytes was performed with antibodies for P2Y 12 receptor, MBP, NG2 . For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (c) Rat cerebellar organotypic cultures were analyzed by double immunofluorescence and confocal microscopy for intra1 (red) and c-ter (red), highlighting different structures (see also insets), and MBP (green).

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in rat brain tissue. Double immunofluorescence and confocal analysis was performed on sections from rat cerebellum (panels (a), (b), (c), (g), (h), and (i)) and striatum (panels (d), (e), (f), (j), (k), and (l)) with intra1 , intra2 -lots AN01/02/04, intra fl , c-ter (all red), and GFAP (green, inset b1), CD11b (green, insets c1, f1, h1; yellow merged, inset i1; green, panel (k); merged, panel (l)), MBP (yellow merged, inset c2; green, inset e1; green, panels (h) and (i); green, inset j1) antibodies.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Temporal and regional pattern of P2Y 12 expression in SOD1-G93A ALS spinal microglia. (a) Double immunofluorescence and confocal analysis on lumbar spinal cord sections (L3–L5) of wild-type (WT) mice was performed with c-ter antibody (green and yellow, merged and insets), CD11b (left panel, yellow, merged and inset), and CD68 (right panel, red, merged and inset), in both dorsal (DH) and ventral (VH) horns of spinal cord. (b) Double immunofluorescence and confocal analysis on SOD1-G93A lumbar spinal cord sections (L3–L5) at two different stages of ALS disease, that is, 20 weeks, and end stage, was performed with c-ter (green) and CD68 (red) antibodies. (c) Equal amount of total lumbar spinal cord lysates (L3–L5) from WT and SOD1-G93A ( n = 4 for each group) were subjected to Western blotting and immunoreactions with c-ter and CD68 antibodies; anti- β -actin was used for protein normalization. Data represent means ± SEM. Statistical significance was calculated by Student's t -test, * P < 0.05.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in human cortex. Sections from human healthy and SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for the immunoreactive markers c-ter (red, panels (a), (c); insets a1, a2, c1; yellow merged, inset c2), intra1 (red, panels (d), insets d1, d2, d3; yellow merged, panel f, insets f1, f2), MBP (green, panels (b), (c), (e), insets c1, e1; yellow merged, panel (f), inset f1), MHC II (green, inset d3), and integrin α II/ β 3 (green, insets b2, e2; yellow merged, insets c2, f2). The asterisks show decreased P2Y 12 immunoreactivity in proximity to MS lesion.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Regional distribution of P2Y 12 in proximity to MS lesions. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for c-ter (red) and MHC II (green) immunoreactivity. In proximity to the demyelinating active cortical lesion expressing augmented positivity for MHC II, microglia gradually lose immunoreactivity for c-ter antibody. Microglia express differential immunoreactivity in the four chosen areas which are found inside (circled b-c) and around (circled a–d) a lesion.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Draw of microglial marker expression as a function of activation. Branched microglia are represented in blue and activated microglia in red. Iba1 , CD11b, and MHC II are mostly expressed in microglia throughout the different morphological states and their expression increases during activation (light blue to red color). P2Y 12 c-ter (light blue) and CD68 (red) are expressed, respectively, in branched or roundish/activated microglia.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Marker, Expressing, Activation Assay

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Primary antibodies employed in the study.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques:

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 antibodies validation and RT-PCR analysis. (a) Scheme of human P2RY 12 gene location, transcript variants [ , ], and protein structure, with amino acid epitopes recognized by the used antibodies and highlighted in color ( intra1 , intra2 , and intra fl , red circle; c-ter , green oval). Species conservation for each epitope was calculated by using BLAT tool of UCSC genome browser . (b) Total protein extracts from SH-SY5Y or HEK293 cells expressing Myc-tagged P2Y 12 receptor were subjected to Western blot analysis with the indicated antibodies. (c) Total protein extracted from human, rat and mouse brain, from primary mouse microglia (mMG) and rat oligodendrocyte (OL) cultures were subjected to Western blot analysis with the indicated antibodies. For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (d) RT-PCR using primers specific for P2Y 12 mRNA was performed on total RNA from rat microglia (rMG) and OL. Control lanes show RT-PCR performed without reverse transcriptase enzyme.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

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

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in dissociated and organotypic primary cultures. (a) Mouse primary cortical microglia were subjected to immunofluorescence and confocal analysis with phalloidin (green, merged field) and P2Y 12 receptor antibodies (red, insets and merged) and Hoechst (white, merged). Scale bars in insets: 20 μ m. (b) Double immunofluorescence and confocal analysis of primary rat mature (OL) and precursor (OPC) oligodendrocytes was performed with antibodies for P2Y 12 receptor, MBP, NG2 . For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (c) Rat cerebellar organotypic cultures were analyzed by double immunofluorescence and confocal microscopy for intra1 (red) and c-ter (red), highlighting different structures (see also insets), and MBP (green).

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in rat brain tissue. Double immunofluorescence and confocal analysis was performed on sections from rat cerebellum (panels (a), (b), (c), (g), (h), and (i)) and striatum (panels (d), (e), (f), (j), (k), and (l)) with intra1 , intra2 -lots AN01/02/04, intra fl , c-ter (all red), and GFAP (green, inset b1), CD11b (green, insets c1, f1, h1; yellow merged, inset i1; green, panel (k); merged, panel (l)), MBP (yellow merged, inset c2; green, inset e1; green, panels (h) and (i); green, inset j1) antibodies.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Temporal and regional pattern of P2Y 12 expression in SOD1-G93A ALS spinal microglia. (a) Double immunofluorescence and confocal analysis on lumbar spinal cord sections (L3–L5) of wild-type (WT) mice was performed with c-ter antibody (green and yellow, merged and insets), CD11b (left panel, yellow, merged and inset), and CD68 (right panel, red, merged and inset), in both dorsal (DH) and ventral (VH) horns of spinal cord. (b) Double immunofluorescence and confocal analysis on SOD1-G93A lumbar spinal cord sections (L3–L5) at two different stages of ALS disease, that is, 20 weeks, and end stage, was performed with c-ter (green) and CD68 (red) antibodies. (c) Equal amount of total lumbar spinal cord lysates (L3–L5) from WT and SOD1-G93A ( n = 4 for each group) were subjected to Western blotting and immunoreactions with c-ter and CD68 antibodies; anti- β -actin was used for protein normalization. Data represent means ± SEM. Statistical significance was calculated by Student's t -test, * P < 0.05.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: P2Y 12 receptor in human cortex. Sections from human healthy and SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for the immunoreactive markers c-ter (red, panels (a), (c); insets a1, a2, c1; yellow merged, inset c2), intra1 (red, panels (d), insets d1, d2, d3; yellow merged, panel f, insets f1, f2), MBP (green, panels (b), (c), (e), insets c1, e1; yellow merged, panel (f), inset f1), MHC II (green, inset d3), and integrin α II/ β 3 (green, insets b2, e2; yellow merged, insets c2, f2). The asterisks show decreased P2Y 12 immunoreactivity in proximity to MS lesion.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Regional distribution of P2Y 12 in proximity to MS lesions. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for c-ter (red) and MHC II (green) immunoreactivity. In proximity to the demyelinating active cortical lesion expressing augmented positivity for MHC II, microglia gradually lose immunoreactivity for c-ter antibody. Microglia express differential immunoreactivity in the four chosen areas which are found inside (circled b-c) and around (circled a–d) a lesion.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing

Journal: Mediators of Inflammation

Article Title: P2Y 12 Receptor on the Verge of a Neuroinflammatory Breakdown

doi: 10.1155/2014/975849

Figure Lengend Snippet: Draw of microglial marker expression as a function of activation. Branched microglia are represented in blue and activated microglia in red. Iba1 , CD11b, and MHC II are mostly expressed in microglia throughout the different morphological states and their expression increases during activation (light blue to red color). P2Y 12 c-ter (light blue) and CD68 (red) are expressed, respectively, in branched or roundish/activated microglia.

Article Snippet: P2Y 12 receptor ( intra2 ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 200–300 , Alomone.

Techniques: Marker, Expressing, Activation Assay