rabbit anti girk2  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti girk2
    Transplantation site influences distribution of A9- and A10-like dopamine neurons within VM grafts. Percentages of <t>Girk2-ir/TH-ir</t> and Calbindin-ir/TH-ir neurons in the periphery and in the centre of E12 (A) and E14 grafts (B) at 6 weeks post-transplantation. Note the decrease in the percentage of Girk2-ir/TH-ir neurons in the periphery of grafts in HPC compared to grafts in other brain regions. The presence of A10 dopamine innervation of the N.Acc significantly increased the percentage of A10-like neurons in the periphery of the graft compared to the graft core in E14 group. Columns depict group means; error bars illustrate ± SEM; significance levels: *p
    Rabbit Anti Girk2, 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
    https://www.bioz.com/result/rabbit anti girk2/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
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    Images

    1) Product Images from "Transplantation site influences the phenotypic differentiation of dopamine neurons in ventral mesencephalic grafts in Parkinsonian rats"

    Article Title: Transplantation site influences the phenotypic differentiation of dopamine neurons in ventral mesencephalic grafts in Parkinsonian rats

    Journal: Experimental Neurology

    doi: 10.1016/j.expneurol.2017.01.010

    Transplantation site influences distribution of A9- and A10-like dopamine neurons within VM grafts. Percentages of Girk2-ir/TH-ir and Calbindin-ir/TH-ir neurons in the periphery and in the centre of E12 (A) and E14 grafts (B) at 6 weeks post-transplantation. Note the decrease in the percentage of Girk2-ir/TH-ir neurons in the periphery of grafts in HPC compared to grafts in other brain regions. The presence of A10 dopamine innervation of the N.Acc significantly increased the percentage of A10-like neurons in the periphery of the graft compared to the graft core in E14 group. Columns depict group means; error bars illustrate ± SEM; significance levels: *p
    Figure Legend Snippet: Transplantation site influences distribution of A9- and A10-like dopamine neurons within VM grafts. Percentages of Girk2-ir/TH-ir and Calbindin-ir/TH-ir neurons in the periphery and in the centre of E12 (A) and E14 grafts (B) at 6 weeks post-transplantation. Note the decrease in the percentage of Girk2-ir/TH-ir neurons in the periphery of grafts in HPC compared to grafts in other brain regions. The presence of A10 dopamine innervation of the N.Acc significantly increased the percentage of A10-like neurons in the periphery of the graft compared to the graft core in E14 group. Columns depict group means; error bars illustrate ± SEM; significance levels: *p

    Techniques Used: Transplantation Assay

    Transplantation site influences A9-like dopamine neuron specification in VM grafts. Coronal sections through E12 VM grafts illustrating TH-ir (green) neurons co-expressing Girk2 (red: A–D) or Calbindin (red: E–H) in grafts in the dSTR (A, E), N.Acc (B, F), PFC (C, G) and HPC (D, H). Note the increase in the Girk2-ir/TH-ir neuron population in grafts in the dSTR compared to other grafts. (A′ and F′) High magnification images from (A and F), illustrating the co-localisation of Girk2 (A′) and Calbindin (F′) with TH and morphology of double labelled neurons within the transplant. (I) Total number of Girk2-ir/TH-ir neurons and (J) Calbindin-ir/TH-ir neurons within the grafts at each donor age and transplantation site. (K) Quantification of the proportion of Girk2-ir/TH-ir neurons and (L) Calbindin-ir/TH-ir neurons out of total TH-ir neurons within the grafts. The presence of targeted midbrain innervation of the transplantation site significantly increased the number and proportion of A9-like neurons in the grafts. Scale bars: 100 μm (A–H) and 25 μm (A′ and F′). Columns depict group means; error bars illustrate ± SEM; significance levels: *p
    Figure Legend Snippet: Transplantation site influences A9-like dopamine neuron specification in VM grafts. Coronal sections through E12 VM grafts illustrating TH-ir (green) neurons co-expressing Girk2 (red: A–D) or Calbindin (red: E–H) in grafts in the dSTR (A, E), N.Acc (B, F), PFC (C, G) and HPC (D, H). Note the increase in the Girk2-ir/TH-ir neuron population in grafts in the dSTR compared to other grafts. (A′ and F′) High magnification images from (A and F), illustrating the co-localisation of Girk2 (A′) and Calbindin (F′) with TH and morphology of double labelled neurons within the transplant. (I) Total number of Girk2-ir/TH-ir neurons and (J) Calbindin-ir/TH-ir neurons within the grafts at each donor age and transplantation site. (K) Quantification of the proportion of Girk2-ir/TH-ir neurons and (L) Calbindin-ir/TH-ir neurons out of total TH-ir neurons within the grafts. The presence of targeted midbrain innervation of the transplantation site significantly increased the number and proportion of A9-like neurons in the grafts. Scale bars: 100 μm (A–H) and 25 μm (A′ and F′). Columns depict group means; error bars illustrate ± SEM; significance levels: *p

    Techniques Used: Transplantation Assay, Expressing

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    Alomone Labs rabbit anti girk2
    Transplantation site influences distribution of A9- and A10-like dopamine neurons within VM grafts. Percentages of <t>Girk2-ir/TH-ir</t> and Calbindin-ir/TH-ir neurons in the periphery and in the centre of E12 (A) and E14 grafts (B) at 6 weeks post-transplantation. Note the decrease in the percentage of Girk2-ir/TH-ir neurons in the periphery of grafts in HPC compared to grafts in other brain regions. The presence of A10 dopamine innervation of the N.Acc significantly increased the percentage of A10-like neurons in the periphery of the graft compared to the graft core in E14 group. Columns depict group means; error bars illustrate ± SEM; significance levels: *p
    Rabbit Anti Girk2, 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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    Alomone Labs kt 5823
    Effects of BPA on [Ca 2+ ] i oscillations through a PKG-mediated mechanism. ( A ) Low-glucose–induced [Ca 2+ ] i oscillations blocked by 1 nM BPA. ( B ) Frequency of [Ca 2+ ] i oscillations were not reduced by BPA in an islet from the same preparation and maintained in the same conditions but pretreated with and exposed to the PKG inhibitor <t>KT-5823</t> (1 μM). ( C ) Mean frequency values of 0.5 mM glucose before application of either BPA (G1) or E 2 (G2), in the presence of 1 nM BPA or 1 nM E 2 , as in ( A ) , or in the presence of 1 μM KT-5823 plus 0.5 mM glucose (KT); KT plus 1 nM BPA (BPA + KT), and KT plus 1 nM 17β-E 2 (E 2 + KT) as in ( B ). Results are representative of at least 12 cells from nine different islets, expressed as mean ± SE. * p
    Kt 5823, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    alomone labs mouse recombinant il 6
    Bile duct staining with CK19 (A–C) and cell proliferation analysis by Ki-67 staining (D, E) were performed 2 weeks after BDL mice. A group of mice received recombinant mouse <t>IL-6</t> (IL-6) or the vehicle only (vehicle) for both sham and BDL surgeries. (A) Representative CK19 staining image for each group. Scale bar: 50 μm. (D, E) Count of CK19 + duct (D; with visible lumen) and nonduct (E; no lumen) per portal area in a blinded manner. The following were the number of animals per group: C57BL/6 sham (7), C57BL/6 sham + IL-6 (3), C57BL/6 BDL (9), C57BL/6 BDL + IL-6 (3), A2bAR −/− sham (7), A2bAR −/− sham + IL-6 (3), A2bAR −/− BDL (13), and A2bAR −/− BDL + IL-6 (3). Between 3 and 5 100× magnification fields were count per animal. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to A2bAR −/− BDL vehicle. (D) Representative image of Ki-67 staining for BDL-operated animal with or without recmIL-6 injection. Scale bar: 50 μm. (E) Analysis of the percent of Ki-67 + pixels on five different 100× magnification fields for three animals per group. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to C57BL/6 BDL IL-6.
    Mouse Recombinant Il 6, supplied by alomone labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Transplantation site influences distribution of A9- and A10-like dopamine neurons within VM grafts. Percentages of Girk2-ir/TH-ir and Calbindin-ir/TH-ir neurons in the periphery and in the centre of E12 (A) and E14 grafts (B) at 6 weeks post-transplantation. Note the decrease in the percentage of Girk2-ir/TH-ir neurons in the periphery of grafts in HPC compared to grafts in other brain regions. The presence of A10 dopamine innervation of the N.Acc significantly increased the percentage of A10-like neurons in the periphery of the graft compared to the graft core in E14 group. Columns depict group means; error bars illustrate ± SEM; significance levels: *p

    Journal: Experimental Neurology

    Article Title: Transplantation site influences the phenotypic differentiation of dopamine neurons in ventral mesencephalic grafts in Parkinsonian rats

    doi: 10.1016/j.expneurol.2017.01.010

    Figure Lengend Snippet: Transplantation site influences distribution of A9- and A10-like dopamine neurons within VM grafts. Percentages of Girk2-ir/TH-ir and Calbindin-ir/TH-ir neurons in the periphery and in the centre of E12 (A) and E14 grafts (B) at 6 weeks post-transplantation. Note the decrease in the percentage of Girk2-ir/TH-ir neurons in the periphery of grafts in HPC compared to grafts in other brain regions. The presence of A10 dopamine innervation of the N.Acc significantly increased the percentage of A10-like neurons in the periphery of the graft compared to the graft core in E14 group. Columns depict group means; error bars illustrate ± SEM; significance levels: *p

    Article Snippet: The primary antibodies and dilution factors used in either bright-field or fluorescent IHC were as follows: mouse anti-NeuN (1:1000; Millipore, Feltham, UK), rabbit anti-TH (1:2000; Millipore), mouse anti-TH (1:1000; Millipore), rabbit anti-Girk2 (1:250; Alomone Labs, Jerusalem, Israel), and mouse anti-Calbindin (1:10,000; Sigma-Aldrich).

    Techniques: Transplantation Assay

    Transplantation site influences A9-like dopamine neuron specification in VM grafts. Coronal sections through E12 VM grafts illustrating TH-ir (green) neurons co-expressing Girk2 (red: A–D) or Calbindin (red: E–H) in grafts in the dSTR (A, E), N.Acc (B, F), PFC (C, G) and HPC (D, H). Note the increase in the Girk2-ir/TH-ir neuron population in grafts in the dSTR compared to other grafts. (A′ and F′) High magnification images from (A and F), illustrating the co-localisation of Girk2 (A′) and Calbindin (F′) with TH and morphology of double labelled neurons within the transplant. (I) Total number of Girk2-ir/TH-ir neurons and (J) Calbindin-ir/TH-ir neurons within the grafts at each donor age and transplantation site. (K) Quantification of the proportion of Girk2-ir/TH-ir neurons and (L) Calbindin-ir/TH-ir neurons out of total TH-ir neurons within the grafts. The presence of targeted midbrain innervation of the transplantation site significantly increased the number and proportion of A9-like neurons in the grafts. Scale bars: 100 μm (A–H) and 25 μm (A′ and F′). Columns depict group means; error bars illustrate ± SEM; significance levels: *p

    Journal: Experimental Neurology

    Article Title: Transplantation site influences the phenotypic differentiation of dopamine neurons in ventral mesencephalic grafts in Parkinsonian rats

    doi: 10.1016/j.expneurol.2017.01.010

    Figure Lengend Snippet: Transplantation site influences A9-like dopamine neuron specification in VM grafts. Coronal sections through E12 VM grafts illustrating TH-ir (green) neurons co-expressing Girk2 (red: A–D) or Calbindin (red: E–H) in grafts in the dSTR (A, E), N.Acc (B, F), PFC (C, G) and HPC (D, H). Note the increase in the Girk2-ir/TH-ir neuron population in grafts in the dSTR compared to other grafts. (A′ and F′) High magnification images from (A and F), illustrating the co-localisation of Girk2 (A′) and Calbindin (F′) with TH and morphology of double labelled neurons within the transplant. (I) Total number of Girk2-ir/TH-ir neurons and (J) Calbindin-ir/TH-ir neurons within the grafts at each donor age and transplantation site. (K) Quantification of the proportion of Girk2-ir/TH-ir neurons and (L) Calbindin-ir/TH-ir neurons out of total TH-ir neurons within the grafts. The presence of targeted midbrain innervation of the transplantation site significantly increased the number and proportion of A9-like neurons in the grafts. Scale bars: 100 μm (A–H) and 25 μm (A′ and F′). Columns depict group means; error bars illustrate ± SEM; significance levels: *p

    Article Snippet: The primary antibodies and dilution factors used in either bright-field or fluorescent IHC were as follows: mouse anti-NeuN (1:1000; Millipore, Feltham, UK), rabbit anti-TH (1:2000; Millipore), mouse anti-TH (1:1000; Millipore), rabbit anti-Girk2 (1:250; Alomone Labs, Jerusalem, Israel), and mouse anti-Calbindin (1:10,000; Sigma-Aldrich).

    Techniques: Transplantation Assay, Expressing

    Effects of BPA on [Ca 2+ ] i oscillations through a PKG-mediated mechanism. ( A ) Low-glucose–induced [Ca 2+ ] i oscillations blocked by 1 nM BPA. ( B ) Frequency of [Ca 2+ ] i oscillations were not reduced by BPA in an islet from the same preparation and maintained in the same conditions but pretreated with and exposed to the PKG inhibitor KT-5823 (1 μM). ( C ) Mean frequency values of 0.5 mM glucose before application of either BPA (G1) or E 2 (G2), in the presence of 1 nM BPA or 1 nM E 2 , as in ( A ) , or in the presence of 1 μM KT-5823 plus 0.5 mM glucose (KT); KT plus 1 nM BPA (BPA + KT), and KT plus 1 nM 17β-E 2 (E 2 + KT) as in ( B ). Results are representative of at least 12 cells from nine different islets, expressed as mean ± SE. * p

    Journal: Environmental Health Perspectives

    Article Title: Low Doses of Bisphenol A and Diethylstilbestrol Impair Ca2+ Signals in Pancreatic ?-Cells through a Nonclassical Membrane Estrogen Receptor within Intact Islets of Langerhans

    doi: 10.1289/ehp.8002

    Figure Lengend Snippet: Effects of BPA on [Ca 2+ ] i oscillations through a PKG-mediated mechanism. ( A ) Low-glucose–induced [Ca 2+ ] i oscillations blocked by 1 nM BPA. ( B ) Frequency of [Ca 2+ ] i oscillations were not reduced by BPA in an islet from the same preparation and maintained in the same conditions but pretreated with and exposed to the PKG inhibitor KT-5823 (1 μM). ( C ) Mean frequency values of 0.5 mM glucose before application of either BPA (G1) or E 2 (G2), in the presence of 1 nM BPA or 1 nM E 2 , as in ( A ) , or in the presence of 1 μM KT-5823 plus 0.5 mM glucose (KT); KT plus 1 nM BPA (BPA + KT), and KT plus 1 nM 17β-E 2 (E 2 + KT) as in ( B ). Results are representative of at least 12 cells from nine different islets, expressed as mean ± SE. * p

    Article Snippet: Thus, KT-5823 does not alter [Ca2+ ]i oscillations but prevents the suppression of low-glucose–induced [Ca2+ ]i oscillations by BPA, indicating that BPA’s effect is exerted by a cGMP/PKG-mediated mechanism, as has been demonstrated for the natural hormone 17β-E2 ( ).

    Techniques:

    Effects of 8Br-cGMP on EDCs and E 2 via PKG. ( A ) Exposure to 10 μM 8Br-cGMP dramatically reduces the frequency of low-glucose–induced [Ca 2+ ] i oscillations. ( B ) After incubation with the specific PKG inhibitor KT-5823 (1 μM), 8Br-cGMP fails to evoke the marked reduction in [Ca 2+ ] i oscillations shown in ( A ). ( C ) Mean frequency values collected in the presence of 0.5 mM glucose (G), 8Br-cGMP plus 0.5 mM glucose (8Br-cGMP), KT-5823 (KT), and 8Br-cGMP plus KT-5823 (8Br-cGMP + KT). Results are representative of at least five cells in four different islets, expressed as mean ± SE.

    Journal: Environmental Health Perspectives

    Article Title: Low Doses of Bisphenol A and Diethylstilbestrol Impair Ca2+ Signals in Pancreatic ?-Cells through a Nonclassical Membrane Estrogen Receptor within Intact Islets of Langerhans

    doi: 10.1289/ehp.8002

    Figure Lengend Snippet: Effects of 8Br-cGMP on EDCs and E 2 via PKG. ( A ) Exposure to 10 μM 8Br-cGMP dramatically reduces the frequency of low-glucose–induced [Ca 2+ ] i oscillations. ( B ) After incubation with the specific PKG inhibitor KT-5823 (1 μM), 8Br-cGMP fails to evoke the marked reduction in [Ca 2+ ] i oscillations shown in ( A ). ( C ) Mean frequency values collected in the presence of 0.5 mM glucose (G), 8Br-cGMP plus 0.5 mM glucose (8Br-cGMP), KT-5823 (KT), and 8Br-cGMP plus KT-5823 (8Br-cGMP + KT). Results are representative of at least five cells in four different islets, expressed as mean ± SE.

    Article Snippet: Thus, KT-5823 does not alter [Ca2+ ]i oscillations but prevents the suppression of low-glucose–induced [Ca2+ ]i oscillations by BPA, indicating that BPA’s effect is exerted by a cGMP/PKG-mediated mechanism, as has been demonstrated for the natural hormone 17β-E2 ( ).

    Techniques: Incubation

    Bile duct staining with CK19 (A–C) and cell proliferation analysis by Ki-67 staining (D, E) were performed 2 weeks after BDL mice. A group of mice received recombinant mouse IL-6 (IL-6) or the vehicle only (vehicle) for both sham and BDL surgeries. (A) Representative CK19 staining image for each group. Scale bar: 50 μm. (D, E) Count of CK19 + duct (D; with visible lumen) and nonduct (E; no lumen) per portal area in a blinded manner. The following were the number of animals per group: C57BL/6 sham (7), C57BL/6 sham + IL-6 (3), C57BL/6 BDL (9), C57BL/6 BDL + IL-6 (3), A2bAR −/− sham (7), A2bAR −/− sham + IL-6 (3), A2bAR −/− BDL (13), and A2bAR −/− BDL + IL-6 (3). Between 3 and 5 100× magnification fields were count per animal. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to A2bAR −/− BDL vehicle. (D) Representative image of Ki-67 staining for BDL-operated animal with or without recmIL-6 injection. Scale bar: 50 μm. (E) Analysis of the percent of Ki-67 + pixels on five different 100× magnification fields for three animals per group. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to C57BL/6 BDL IL-6.

    Journal: Gene Expression

    Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

    doi: 10.3727/105221617X15042723767876

    Figure Lengend Snippet: Bile duct staining with CK19 (A–C) and cell proliferation analysis by Ki-67 staining (D, E) were performed 2 weeks after BDL mice. A group of mice received recombinant mouse IL-6 (IL-6) or the vehicle only (vehicle) for both sham and BDL surgeries. (A) Representative CK19 staining image for each group. Scale bar: 50 μm. (D, E) Count of CK19 + duct (D; with visible lumen) and nonduct (E; no lumen) per portal area in a blinded manner. The following were the number of animals per group: C57BL/6 sham (7), C57BL/6 sham + IL-6 (3), C57BL/6 BDL (9), C57BL/6 BDL + IL-6 (3), A2bAR −/− sham (7), A2bAR −/− sham + IL-6 (3), A2bAR −/− BDL (13), and A2bAR −/− BDL + IL-6 (3). Between 3 and 5 100× magnification fields were count per animal. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to A2bAR −/− BDL vehicle. (D) Representative image of Ki-67 staining for BDL-operated animal with or without recmIL-6 injection. Scale bar: 50 μm. (E) Analysis of the percent of Ki-67 + pixels on five different 100× magnification fields for three animals per group. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to C57BL/6 BDL IL-6.

    Article Snippet: For some experiments, A2bAR−/− and C57BL/6 mice were injected subcutaneously with 250 ng/g of body weight of mouse recombinant IL-6 (recmIL-6; BioLegend, San Diego, CA, USA) or vehicle 15 min prior to the surgery and once a week for the duration of the experiments.

    Techniques: Staining, Mouse Assay, Recombinant, Injection

    Second messenger alterations and control of IL-6 release mediated by adenosine/A2bAR. Intracellular cAMP concentrations (A, B) were analyzed by ELISA. H69 cells were stimulated with 100 μM adenosine for 2 h in 96-well plates (5,000 cells per well) in H69 complete media in the presence of A2bAR antagonist (A) MRS-1754 (1 μM; n = 3, performed in triplicates) or (B) 72 h after transfection with an A2bAR-specific siRNA ( n = 4, performed in triplicate). #Significant when compared to the untreated cells; *significant when compared to vehicle (A) or nontarget siRNA (B). (C, D) Intracellular Ca 2+ mobilization was observed by confocal microscopy. Glass coverslip-plated, fluo-4/AM-loaded H69 cells were perifused with HEPES buffer alone or containing 500 μM adenosine. ATP (100 μM) was used as a positive control. Changes in fluorescence intensity were recorded with a Zeiss 510 confocal microscope. (C) Relative fluorescence intensity of individual cells; 29 individual cells were analyzed. (D) Representative image of the dye intensity variation during the course of the experiment. Scale bar: 50 μm. (E) H69 cells were stimulated by 100 μM of adenosine for 2 h in six-well plates (150,000 cells per well) in H69 complete media. RNA was extracted, and IL-6 mRNA expression was determined by qPCR. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation ( n = 3 for cAMPs-RP and n = 7 for BAPTA/AM). (F) H69 cells were stimulated by 100 μM adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate). #Significant when compared to unstimulated cells; *significant when compared to vehicle. (G) H69 cells were stimulated by 100 μM of 8-pCPT-2-O-Me-cAMP-AM (8-pCPT) or adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate).

    Journal: Gene Expression

    Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

    doi: 10.3727/105221617X15042723767876

    Figure Lengend Snippet: Second messenger alterations and control of IL-6 release mediated by adenosine/A2bAR. Intracellular cAMP concentrations (A, B) were analyzed by ELISA. H69 cells were stimulated with 100 μM adenosine for 2 h in 96-well plates (5,000 cells per well) in H69 complete media in the presence of A2bAR antagonist (A) MRS-1754 (1 μM; n = 3, performed in triplicates) or (B) 72 h after transfection with an A2bAR-specific siRNA ( n = 4, performed in triplicate). #Significant when compared to the untreated cells; *significant when compared to vehicle (A) or nontarget siRNA (B). (C, D) Intracellular Ca 2+ mobilization was observed by confocal microscopy. Glass coverslip-plated, fluo-4/AM-loaded H69 cells were perifused with HEPES buffer alone or containing 500 μM adenosine. ATP (100 μM) was used as a positive control. Changes in fluorescence intensity were recorded with a Zeiss 510 confocal microscope. (C) Relative fluorescence intensity of individual cells; 29 individual cells were analyzed. (D) Representative image of the dye intensity variation during the course of the experiment. Scale bar: 50 μm. (E) H69 cells were stimulated by 100 μM of adenosine for 2 h in six-well plates (150,000 cells per well) in H69 complete media. RNA was extracted, and IL-6 mRNA expression was determined by qPCR. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation ( n = 3 for cAMPs-RP and n = 7 for BAPTA/AM). (F) H69 cells were stimulated by 100 μM adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate). #Significant when compared to unstimulated cells; *significant when compared to vehicle. (G) H69 cells were stimulated by 100 μM of 8-pCPT-2-O-Me-cAMP-AM (8-pCPT) or adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate).

    Article Snippet: For some experiments, A2bAR−/− and C57BL/6 mice were injected subcutaneously with 250 ng/g of body weight of mouse recombinant IL-6 (recmIL-6; BioLegend, San Diego, CA, USA) or vehicle 15 min prior to the surgery and once a week for the duration of the experiments.

    Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Confocal Microscopy, Positive Control, Fluorescence, Microscopy, Expressing, Real-time Polymerase Chain Reaction

    Effect of A2bAR knockout on survival and fibrosis after bile duct ligation (BDL). BDL was performed on 10- to 12-week-old male C57BL/6 or A2bAR −/− mice. (A–D) A group of mice received weekly subcutaneous injection of recombinant mouse IL-6 (IL-6; n = 13 for both background), and the other group received vehicle only ( n = 8 for both background). (A) Kaplan–Meier survival graph. The black line represents control C57BL/6 mice, and the blue line corresponds to A2bAR −/− mice. Full lines are vehicle only, and dotted lines represent mice that received recmIL-6 injections. *Significant when compared to C57BL/6 mice; #significant when compared to vehicle injection. (B) Mason trichrome staining representative of each group at the median time of survival. Scale bar: 100 μm. (C) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (D) Bile infarcts were counted on a blinded manner on low-power field hematoxylin and eosin staining of each individual mouse. (E–H) BDL or sham operation was performed on 10- to 12-week-old male C57BL/6 ( n = 9 and 7, respectively) or A2bAR −/− mice ( n = 13 and 7, respectively). Mice were sacrificed 2 weeks after surgery. (E) Representative picture of hematoxylin and eosin (H E), Pico Sirius red (PSR), and Mason trichrome staining for each group of mice. Scale bar: 100 μm. (F) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (G) Total liver RNA was extracted, and relative IL-6 mRNA was assessed by qPCR. GAPDH -specific probe was used as control. C57BL/6 and A2bAR −/− sham operated levels were, respectively, used as reference for the BDL group. (H) Serum was obtained from sham and BDL-operated mice, and total serum IL-6 was assessed by ELISA.

    Journal: Gene Expression

    Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

    doi: 10.3727/105221617X15042723767876

    Figure Lengend Snippet: Effect of A2bAR knockout on survival and fibrosis after bile duct ligation (BDL). BDL was performed on 10- to 12-week-old male C57BL/6 or A2bAR −/− mice. (A–D) A group of mice received weekly subcutaneous injection of recombinant mouse IL-6 (IL-6; n = 13 for both background), and the other group received vehicle only ( n = 8 for both background). (A) Kaplan–Meier survival graph. The black line represents control C57BL/6 mice, and the blue line corresponds to A2bAR −/− mice. Full lines are vehicle only, and dotted lines represent mice that received recmIL-6 injections. *Significant when compared to C57BL/6 mice; #significant when compared to vehicle injection. (B) Mason trichrome staining representative of each group at the median time of survival. Scale bar: 100 μm. (C) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (D) Bile infarcts were counted on a blinded manner on low-power field hematoxylin and eosin staining of each individual mouse. (E–H) BDL or sham operation was performed on 10- to 12-week-old male C57BL/6 ( n = 9 and 7, respectively) or A2bAR −/− mice ( n = 13 and 7, respectively). Mice were sacrificed 2 weeks after surgery. (E) Representative picture of hematoxylin and eosin (H E), Pico Sirius red (PSR), and Mason trichrome staining for each group of mice. Scale bar: 100 μm. (F) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (G) Total liver RNA was extracted, and relative IL-6 mRNA was assessed by qPCR. GAPDH -specific probe was used as control. C57BL/6 and A2bAR −/− sham operated levels were, respectively, used as reference for the BDL group. (H) Serum was obtained from sham and BDL-operated mice, and total serum IL-6 was assessed by ELISA.

    Article Snippet: For some experiments, A2bAR−/− and C57BL/6 mice were injected subcutaneously with 250 ng/g of body weight of mouse recombinant IL-6 (recmIL-6; BioLegend, San Diego, CA, USA) or vehicle 15 min prior to the surgery and once a week for the duration of the experiments.

    Techniques: Knock-Out, Ligation, Mouse Assay, Injection, Recombinant, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay