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

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Functional expression and intracellular signaling of UTP-sensitive P2Y receptors in theca-interstitial cells

doi: 10.1186/1477-7827-8-88

Figure Lengend Snippet: p2y2r, p2y4r , and p2y6r expression in mouse theca cells . (A) RNA from cultured theca cells (1), whole ovary (2), heart (3), and brain (4) was reverse transcribed and amplified by PCR for p2Y2, p2Y4, p2Y6, cyp11A, cyp 17A, star, fshr, and β-actin using specific oligonucleotides to detect transcript expression. Controls in lanes (5) and (6) correspond to a PCR reaction of a theca cell RNA sample without reverse transcriptase and to the reaction mix without a cDNA template, respectively. Amplicon length is indicated in base pairs (bp). Each amplification assay was repeated in 3-5 independent cultures. (B) CREB phosphorylation evaluated by Western blot from TIC cultures in basal conditions or stimulated for 10 min by either 2 IU hCG or 1 ng/ml FSH. The 43-kDa band was analyzed by densitometry, and the result (mean ± S.E.M) of three determinations from independent cultures was plotted (*p < 0.01). PARP protein was used as gel loading control. (C) P2Y2 and P2Y6 receptors detected by Western blot (WB), from freshly isolated TIC homogenates directly (P2Y2) or after immunoprecipitation (IP) (P2Y6).

Article Snippet: TIC (1 × 10 6 ) were scraped in ice-cold TNTE buffer (Tris-HCl pH 7.4,150 mM NaCl, 50 mM and 0.5 mM EDTA) containing 5% Triton X-100 and a protease inhibitor cocktail (Roche Co., Basel, Switzerland); the lysate was centrifuged for 10 min at 14,000 rpm at 4°C, and the soluble fraction was incubated overnight with 3 μl of anti-P2Y6 antibody (Alomone, Jerusalem, Israel).

Techniques: Expressing, Cell Culture, Reverse Transcription, Amplification, Phospho-proteomics, Western Blot, Control, Isolation, Immunoprecipitation