Journal:

Article Title: Adenine nucleotides inhibit recombinant N-type calcium channels via G protein-coupled mechanisms in HEK 293 cells; involvement of the P2Y 13 receptor-type

doi: 10.1038/sj.bjp.0705588

Figure Lengend Snippet: P2Y receptor mRNA expression and immunohistochemistry in HEK 293-N26 cells. (A) Subsequent to total RNA extraction and RT–PCR amplification with primers specific for distinct P2Y receptor cDNA fragments (P2Y13, 25 cycles; P2Y1–P2Y12, 35 cycles), cDNA products were analyzed by agarose gel (1.5%) electrophoresis. A representative gel with ethidium bromide-stained cDNA fragments is shown: P2Y1 (528 bp), P2Y4 (431 bp), P2Y6 (380 bp), P2Y11 (410) and P2Y13 (575 bp). P2Y2 and P2Y12 transcripts were not detectable. (B) Representative fluorescence image of HEK 293-N26 cells after immunocytochemical labeling of P2Y1 (a) and P2Y4 receptors (c), with rabbit anti-P2Y1 and P2Y4 receptor antibodies (scale bar, 20 μm). P2Y2 receptor immunoreactivities (b) could not be detected.

Article Snippet: After fixation, washing with Tris-buffered saline (TBS, 0.05 M; pH 7.6) and blocking with 5% fetal calf serum (FCS), the cells were incubated with the rabbit anti-P2Y receptor antibodies (anti-P2Y 1 : 1 : 1500, SmithKline Beecham Pharmaceuticals, Harlow, Essex, U.K.; anti-P2Y 2 : 1 : 1000, Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, MD, U.S.A.; anti-P2Y 4 : 1 : 1000, Alomone Labs, Jerusalem, Israel) with 0.1% Triton X-100, 5% FCS in TBS for 12 h at 4°C.

Techniques: Expressing, Immunohistochemistry, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Staining, Fluorescence, Labeling

Journal: Purinergic Signalling

Article Title: Purinergic receptors expressed in human skeletal muscle fibres

doi: 10.1007/s11302-011-9279-y

Figure Lengend Snippet: Distribution of purinergic receptors in healthy subjects

Article Snippet: The P2X antibody was highly specific and directed against the epitope corresponding to amino acid residues 382–399 of rat P2X (human 15/18 residues identical); the P2Y 11 antibody was corresponding to amino acid residues 357–373 of human P2Y 11 ; western blots for anti-P2X and anti-P2Y 11 are shown on the Alomone homepage.

Techniques:

Journal: Purinergic Signalling

Article Title: Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

doi: 10.1007/s11302-008-9108-0

Figure Lengend Snippet: Expression of P2Y 1 and P2Y 2 receptors in human anagen hair follicles. a P2Y 1 receptors were found in the outer root sheath ( ORS ) and bulb of anagen hair follicles in longitudinal section but not in the inner root sheath ( IRS ). Scale bar = 40 μm. b In transverse section, P2Y 1 receptors were only seen in the outer root sheath ( ORS ). Scale bar = 40 μm. c P2Y 2 receptors were found in the cortex ( CTX ). Scale bar = 40 μm. d Transverse section of anagen hair follicle: P2Y 2 receptors were seen in the cortex ( CTX ) but not in the central medulla ( M ), inner ( IRS ) or outer root sheaths ( ORS ), or in the surrounding adventitial layer ( A ). Scale bar = 40 μm. e , f Controls: the immunoreaction was abolished after preabsorption of the e P2Y 1 and f P2Y 2 receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. Scale bars = 100 μm

Article Snippet: Polyclonal anti-P2Y 1 and P2Y 2 antibodies were obtained from Alomone Labs (Jerusalem, Israel) and corresponded to the third extracellular loop of the P2Y 1 (AA 242–258) and P2Y 2 receptor (AA 227–244).

Techniques: Expressing

Journal: Purinergic Signalling

Article Title: Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

doi: 10.1007/s11302-008-9108-0

Figure Lengend Snippet: Double labelling of P2Y 1 and P2Y 2 receptors with markers for cellular proliferation, and double labelling of P2X 5 receptors with markers for keratinocyte differentiation in anagen hair follicles. a Double labelling of P2Y 1 receptors ( red ) with Ki-67, a nuclear marker for proliferating cells ( green ), to show that P2Y 1 receptors are found in proliferating basal cells in the outer root sheath ( ORS ) and bulb region of the hair follicle in longitudinal section. Scale bar = 50 μm. b Transverse section: double labelling of P2Y 1 receptors ( red ) with Ki-67, a nuclear marker ( green ), to show that P2Y 1 receptors are found in proliferating cells in the outer root sheath ( ORS ) of the hair follicle. Scale bar = 50 μm. c Longitudinal section of anagen hair follicle through the dermal papilla ( DP ): double labelling of P2Y 2 receptors ( red ) with proliferating cell nuclear antigen (PCNA), a marker for proliferating cells ( green ), showed that P2Y 2 receptors were found in the cortex ( CTX ) and at the edge of the medulla ( M ) but not in the central medulla. P2Y 2 receptors were not found in the matrix ( Ma ), where cells were positive for PCNA. Scale bar = 75 μm. d Longitudinal section of anagen hair follicle: P2Y 2 receptors were absent from the keratinised cuticle ( Cu ) of the hair shaft. PCNA was also found in cells of the outer root sheath ( ORS ). Scale bar = 75 μm. e Double labelling of P2X 5 receptors ( red-brown ) with involucrin, a marker for differentiating cells ( green ). Involucrin was expressed both in the inner root sheath ( IRS ), cortex ( CTX ) and in the outermost edge of the medulla ( M ). P2X 5 receptors were expressed in the inner ( IRS ) and outer root sheaths ( ORS ) and in the medulla ( M ) and matrix cells ( Ma ). There was yellow colocalisation with P2X 5 receptors in the inner root sheath and in cells at the outermost edge of the medulla ( arrow ). The cortex only stained positive for involucrin, not P2X 5 receptors. Scale bar = 50 μm. f Transverse section: double labelling of P2X 5 receptors ( red ) with involucrin ( green ). Involucrin was expressed in the inner root sheath ( IRS ) and cortex ( CTX ) and colocalised ( yellow ) with P2X 5 receptor staining in the inner root sheath ( IRS ). Scale bar = 50 μm

Article Snippet: Polyclonal anti-P2Y 1 and P2Y 2 antibodies were obtained from Alomone Labs (Jerusalem, Israel) and corresponded to the third extracellular loop of the P2Y 1 (AA 242–258) and P2Y 2 receptor (AA 227–244).

Techniques: Marker, Staining

Journal: Biomolecules

Article Title: Gintonin Binds to Reduced LPA4 Receptor Subtype in Human Cortical Neurons in Alzheimer’s Disease Brains

doi: 10.3390/biom15020179

Figure Lengend Snippet: Co-localization of the gintonin (GT)-binding site with the LPA4 receptor subtype and a selective reduction in LPA4 receptor subtype expression levels in patients with AD. ( A ) Representative confocal images of the LPA receptor subtypes (LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, and LPAR6; green) and GT (red) in the cortices of the HCs and patients with AD. Scale bar = 200 μm. The insert images focus on the co-localization between gintonin (GT) and the LAP receptor subtypes. Scale bar = 100 μm. ( B ) Quantitative analysis of the co-localization between the GT and LPA receptor subtypes. ( C – F ) Western blot analysis of the LPA receptor subtypes in brain tissue from the HCs and patients with AD. The results indicate a significant decrease in LPAR4 expression in patients with AD, whereas no significant differences were observed for the other LPA receptor subtypes. Statistical significance was determined using a t -test. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01. Original Western blot images can be found in .

Article Snippet: The primary antibodies used were mouse anti-NeuN (1:500, #MAB377, EMD Millipore, Billerica, MA, USA), rabbit anti-Iba1 (1:500, 019-19741, Wako, Osaka, Japan), FITC-anti-CD11b (1:100, 101206, Bio Legend, San Diego, CA, USA), mouse anti-mouse GFAP (1:500, 3670S, CST, Danvers, MA, USA), rabbit anti-LPAR1 (1:500, ALR-031, Alomone labs, Jerusalem, Israel), rabbit anti-LPAR2 (1:500, ALR-032, Alomone labs), rabbit anti-LPAR3 (1:500, ab23692, Abcam, Cambrighe, UK), rabbit anti-LPAR4 (1:500, ALR-034, Alomone labs), rabbit anti-LPAR5 (1:500, ALR-035, Alomone labs), and rabbit anti-LPAR6 (1:500, ALR-036, Alomone labs).

Techniques: Binding Assay, Expressing, Western Blot

Journal: Biomolecules

Article Title: Gintonin Binds to Reduced LPA4 Receptor Subtype in Human Cortical Neurons in Alzheimer’s Disease Brains

doi: 10.3390/biom15020179

Figure Lengend Snippet: The relationship between LPA4 receptor subtype expression in the cortices of HCs and patients with AD. ( A ) Representative confocal images showing DAPI (blue), LPAR4 (green), and NeuN (red) in the cortices of HCs and patients with AD. Scale bar = 100 μm. ( B ) Quantitative analysis of the co-localization between the LPA4 receptor subtype and NeuN. ( C ) Representative confocal images showing DAPI (blue), LPAR4 (green), and GFAP (red) in the brains of HCs and patients with AD. Scale bar = 100 μm. ( D ) Quantitative analysis of the co-localization between the LPA4 receptor subtype and GFAP. ( E ) Representative confocal images showing DAPI (blue), LPAR4 (green), and Iba1 (red) in the cortices of HCs and patients with AD. Scale bar = 100 μm. ( F ) Quantitative analysis of the co-localization between the LPA receptor subtype and Iba 1. Statistical significance was determined using a t -test. Data are presented as mean ± SEM. * p < 0.05.

Article Snippet: The primary antibodies used were mouse anti-NeuN (1:500, #MAB377, EMD Millipore, Billerica, MA, USA), rabbit anti-Iba1 (1:500, 019-19741, Wako, Osaka, Japan), FITC-anti-CD11b (1:100, 101206, Bio Legend, San Diego, CA, USA), mouse anti-mouse GFAP (1:500, 3670S, CST, Danvers, MA, USA), rabbit anti-LPAR1 (1:500, ALR-031, Alomone labs, Jerusalem, Israel), rabbit anti-LPAR2 (1:500, ALR-032, Alomone labs), rabbit anti-LPAR3 (1:500, ab23692, Abcam, Cambrighe, UK), rabbit anti-LPAR4 (1:500, ALR-034, Alomone labs), rabbit anti-LPAR5 (1:500, ALR-035, Alomone labs), and rabbit anti-LPAR6 (1:500, ALR-036, Alomone labs).

Techniques: Expressing