ngf  (Alomone Labs)


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    Structured Review

    Alomone Labs ngf
    Hypothesis of inhibition mechanism of the <t>NGF-induced</t> neurite extension by chronic irradiation with low-dose-rate 137 <t>Cs-γ</t> rays via Ca 2+ /calmodulin-dependent kinase II activation. Chronic irradiation with low-dose-rate γ rays activates CaMKII in the downstream of the Ca-dependent signal induced by NGF. The production of reactive oxygen species generated by irradiation may be involved in this activation of CaMKII. The CaMK II suppresses NGF-induced neuronal extension by inhibition of the activation of Rac1 downstream of the PI3K-Akt signal. IQGAP1, which is considered to contribute to neurite extension, may be involved in this inhibition of Rac1 activity. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase, PI3K = phosphoinositide 3-kinase, IP3 = inositol trisphosphate, CaM = calmodulin, CaMKII = Ca 2+ /calmodulin-dependent protein kinase II, ROS = reactive oxygen species, IQGAP1 = Ras GTPase-activating-like protein.
    Ngf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation"

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    Journal: Journal of Radiation Research

    doi: 10.1093/jrr/rrx032

    Hypothesis of inhibition mechanism of the NGF-induced neurite extension by chronic irradiation with low-dose-rate 137 Cs-γ rays via Ca 2+ /calmodulin-dependent kinase II activation. Chronic irradiation with low-dose-rate γ rays activates CaMKII in the downstream of the Ca-dependent signal induced by NGF. The production of reactive oxygen species generated by irradiation may be involved in this activation of CaMKII. The CaMK II suppresses NGF-induced neuronal extension by inhibition of the activation of Rac1 downstream of the PI3K-Akt signal. IQGAP1, which is considered to contribute to neurite extension, may be involved in this inhibition of Rac1 activity. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase, PI3K = phosphoinositide 3-kinase, IP3 = inositol trisphosphate, CaM = calmodulin, CaMKII = Ca 2+ /calmodulin-dependent protein kinase II, ROS = reactive oxygen species, IQGAP1 = Ras GTPase-activating-like protein.
    Figure Legend Snippet: Hypothesis of inhibition mechanism of the NGF-induced neurite extension by chronic irradiation with low-dose-rate 137 Cs-γ rays via Ca 2+ /calmodulin-dependent kinase II activation. Chronic irradiation with low-dose-rate γ rays activates CaMKII in the downstream of the Ca-dependent signal induced by NGF. The production of reactive oxygen species generated by irradiation may be involved in this activation of CaMKII. The CaMK II suppresses NGF-induced neuronal extension by inhibition of the activation of Rac1 downstream of the PI3K-Akt signal. IQGAP1, which is considered to contribute to neurite extension, may be involved in this inhibition of Rac1 activity. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase, PI3K = phosphoinositide 3-kinase, IP3 = inositol trisphosphate, CaM = calmodulin, CaMKII = Ca 2+ /calmodulin-dependent protein kinase II, ROS = reactive oxygen species, IQGAP1 = Ras GTPase-activating-like protein.

    Techniques Used: Inhibition, Irradiation, Activation Assay, Generated, Activity Assay, Chick Chorioallantoic Membrane Assay

    Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P
    Figure Legend Snippet: Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Techniques Used: Irradiation

    Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.
    Figure Legend Snippet: Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.

    Techniques Used: Western Blot, Irradiation

    Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P
    Figure Legend Snippet: Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Techniques Used: Inhibition, Activity Assay, Irradiation

    Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P
    Figure Legend Snippet: Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P

    Techniques Used: Irradiation, Activation Assay, Activity Assay

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    Alomone Labs anti nr2b
    Leptin signaling increases pNR2B Y1472 levels and surface expression. ( A ) Representative Western blot of hippocampal neurons treated with leptin (50 nM), PP1 (10 µM), or both for 2 hours. ( B ) Quantification of pNR2B Y1472 intensity normalized to total <t>NR2B</t> intensity (n = 3). ( C ) Representative Western blot of hippocampal protein extracts from P10 wild-type and ob/ob mice pups (wild-type: n = 5; ob/ob : n = 5). ( D ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity and total NR2B intensity normalized to the neuronal marker MAP2B intensity (n = 3). ( E ) Representative Western blot of surface biotinylated hippocampal cultures treated with leptin (50 nM, 2 hours). Biotinylated proteins were affinity purified (AP) with streptavidin magnetic beads. ( F ) Quantification of biotinylated NR2B intensity normalized to NR2B intensity in total lysate (n = 3). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P
    Anti Nr2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit anti nr1
    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding <t>NR1,</t> NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P
    Rabbit Anti Nr1, 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 pc12 cells
    Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in <t>PC12</t> cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P
    Pc12 Cells, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Alomone Labs retigabine
    Protein kinase A (PKA)-dependent regulation of endogenous Kv7.5 currents in cultured HASMCs. ( A ) Representative current traces recorded in a single HASMC (Capacitance = 281 pF) before (i. control) and 5 min after addition of 10 µM <t>retigabine</t> (ii). ( B ) Mean fractional conductance plot calculated from steady-state endogenous Kv7 currents fitted to a Boltzmann distribution (V 0.5 = −40.8 mV, n = 10). ( C ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 5), after 5 min treatment with 1 µM forskolin (open circles, n = 4), and after 5 min treatment with diclofenac (100 µM, open triangles, n = 4). ( D ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 7), after 5 min treatment with 1 nM formoterol (open circles, n = 7), and after 5 min treatment with Kv7 channel blocker XE991 (1 µM) in the presence of 1 nM formoterol (closed triangles, n = 3). ( E ) Representative time-courses of 1 nM formoterol application recorded at −20 mV in a single untreated HASMC (black, Capacitance = 39 pF) and a HASMC pretreated with 10 µM H-89 (red, C = 127 pF). ( F ) Relative formoterol-induced enhancement of the current recorded at −20 mV in untreated HASMCs (black bars, n = 7) and in HASMCs pretreated with H-89 (10 µM for 20 min, red bar, n = 4). * Significant difference from control ( p
    Retigabine, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Leptin signaling increases pNR2B Y1472 levels and surface expression. ( A ) Representative Western blot of hippocampal neurons treated with leptin (50 nM), PP1 (10 µM), or both for 2 hours. ( B ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity (n = 3). ( C ) Representative Western blot of hippocampal protein extracts from P10 wild-type and ob/ob mice pups (wild-type: n = 5; ob/ob : n = 5). ( D ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity and total NR2B intensity normalized to the neuronal marker MAP2B intensity (n = 3). ( E ) Representative Western blot of surface biotinylated hippocampal cultures treated with leptin (50 nM, 2 hours). Biotinylated proteins were affinity purified (AP) with streptavidin magnetic beads. ( F ) Quantification of biotinylated NR2B intensity normalized to NR2B intensity in total lysate (n = 3). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Journal: Endocrinology

    Article Title: Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B

    doi: 10.1210/endocr/bqz030

    Figure Lengend Snippet: Leptin signaling increases pNR2B Y1472 levels and surface expression. ( A ) Representative Western blot of hippocampal neurons treated with leptin (50 nM), PP1 (10 µM), or both for 2 hours. ( B ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity (n = 3). ( C ) Representative Western blot of hippocampal protein extracts from P10 wild-type and ob/ob mice pups (wild-type: n = 5; ob/ob : n = 5). ( D ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity and total NR2B intensity normalized to the neuronal marker MAP2B intensity (n = 3). ( E ) Representative Western blot of surface biotinylated hippocampal cultures treated with leptin (50 nM, 2 hours). Biotinylated proteins were affinity purified (AP) with streptavidin magnetic beads. ( F ) Quantification of biotinylated NR2B intensity normalized to NR2B intensity in total lysate (n = 3). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Article Snippet: Neurons fixed and incubated with an anti-NR2B (1:100, Alomone Labs) ( ) and anti-Flag (1:250, Sigma Aldrich) ( ) antibody for 1 hour and then incubated with the appropriate Alexa Fluor secondary IgG antibody ( ) for 1 hour at room temperature.

    Techniques: Expressing, Western Blot, Mouse Assay, Marker, Affinity Purification, Magnetic Beads

    Leptin-regulated NR2B Y1472 phosphorylation and surface expression is Fyn dependent. ( A ) Representative Western blot of HEK293T cells transfected with NR2B-V5, NR1, LepRb-myc, and either V5-Fyn or V5-DN Fyn and treated with leptin (50 nM, 2 hours). ( B ) Quantification of pNR2B Y1472 intensity normalized to total NR2B-V5 intensity (n = 3). ( C ) Hippocampal neurons were transfected with Clover and EGFP-NR2B-V5 and either V5-Fyn or V5-DN Fyn ± leptin stimulation (50 nM, 2 hours) and live immunostained for surface EGFP–NR2B. Quantification of immunostained EGFP-integrated signal density (n = 15). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Journal: Endocrinology

    Article Title: Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B

    doi: 10.1210/endocr/bqz030

    Figure Lengend Snippet: Leptin-regulated NR2B Y1472 phosphorylation and surface expression is Fyn dependent. ( A ) Representative Western blot of HEK293T cells transfected with NR2B-V5, NR1, LepRb-myc, and either V5-Fyn or V5-DN Fyn and treated with leptin (50 nM, 2 hours). ( B ) Quantification of pNR2B Y1472 intensity normalized to total NR2B-V5 intensity (n = 3). ( C ) Hippocampal neurons were transfected with Clover and EGFP-NR2B-V5 and either V5-Fyn or V5-DN Fyn ± leptin stimulation (50 nM, 2 hours) and live immunostained for surface EGFP–NR2B. Quantification of immunostained EGFP-integrated signal density (n = 15). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Article Snippet: Neurons fixed and incubated with an anti-NR2B (1:100, Alomone Labs) ( ) and anti-Flag (1:250, Sigma Aldrich) ( ) antibody for 1 hour and then incubated with the appropriate Alexa Fluor secondary IgG antibody ( ) for 1 hour at room temperature.

    Techniques: Expressing, Western Blot, Transfection

    LepRb directly interacts with NR2B. ( A ) Schematic of LepRb–BioID experiment with representative Western blot of NR2B-V5 immunoprecipitated from HEK293T cells expressing the designated BioID constructs and NR2B-V5 and NR1-Clover to the right. ( B ) Quantification of IP biotinylated NR2B-V5 intensity normalized to total NR2B-V5 intensity in the same lane (n = 3). ( C ) Schematic of NR2B–BioID experiment with representative Western blot of LepRb-V5 immunoprecipitated from HEK293T cells expressing designated BioID constructs and LepRb-V5 and NR1-Clover. ( D ) Representative Western blot of LepRb-myc immunoprecipitated from HEK293T cells stimulated with leptin (50 nM, 2 hours) and expressing LepRb-myc, NR2B-V5, and NR1-Clover. ( E ) Quantification of coimmunoprecipitated NR2B-V5 intensity normalized to immunoprecipitated LepRb-myc intensity from the same lane (n = 3). ( F ) Representative fluorescent images of hippocampal cultures expressing Flag-LepRb and Clover. Surface Flag-LepRb and endogenous surface NR2B were live immunostained after stimulation with leptin (50 nM, 2 hours). ( G ) Quantification of NR2B/Flag-LepRb puncta colocalization compared to total NR2B puncta. Colocalization experiments were repeated in 2 independent hippocampal culture preparations. All BioID experiments were stimulated with biotin (50 µM) at the time of transfection. All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Journal: Endocrinology

    Article Title: Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B

    doi: 10.1210/endocr/bqz030

    Figure Lengend Snippet: LepRb directly interacts with NR2B. ( A ) Schematic of LepRb–BioID experiment with representative Western blot of NR2B-V5 immunoprecipitated from HEK293T cells expressing the designated BioID constructs and NR2B-V5 and NR1-Clover to the right. ( B ) Quantification of IP biotinylated NR2B-V5 intensity normalized to total NR2B-V5 intensity in the same lane (n = 3). ( C ) Schematic of NR2B–BioID experiment with representative Western blot of LepRb-V5 immunoprecipitated from HEK293T cells expressing designated BioID constructs and LepRb-V5 and NR1-Clover. ( D ) Representative Western blot of LepRb-myc immunoprecipitated from HEK293T cells stimulated with leptin (50 nM, 2 hours) and expressing LepRb-myc, NR2B-V5, and NR1-Clover. ( E ) Quantification of coimmunoprecipitated NR2B-V5 intensity normalized to immunoprecipitated LepRb-myc intensity from the same lane (n = 3). ( F ) Representative fluorescent images of hippocampal cultures expressing Flag-LepRb and Clover. Surface Flag-LepRb and endogenous surface NR2B were live immunostained after stimulation with leptin (50 nM, 2 hours). ( G ) Quantification of NR2B/Flag-LepRb puncta colocalization compared to total NR2B puncta. Colocalization experiments were repeated in 2 independent hippocampal culture preparations. All BioID experiments were stimulated with biotin (50 µM) at the time of transfection. All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Article Snippet: Neurons fixed and incubated with an anti-NR2B (1:100, Alomone Labs) ( ) and anti-Flag (1:250, Sigma Aldrich) ( ) antibody for 1 hour and then incubated with the appropriate Alexa Fluor secondary IgG antibody ( ) for 1 hour at room temperature.

    Techniques: Western Blot, Immunoprecipitation, Expressing, Construct, Transfection

    pNR2B Y1472 is necessary for leptin-stimulated spine formation. ( A ) Representative fluorescent images of hippocampal neurons expressing Clover and EGFP-NR2B-V5 or EGFP-NR2B Y1472F -V5 ± leptin stimulation (50 nM, 2 hours) and live immunostained for surface EGFP-NR2B. White bar = 20 µm. ( B ) Quantification of immunostained EGFP-integrated signal density (n = 23). ( C-E ) Hippocampal neurons that were transfected with a fluorescent Clover-βactin and EGFP-NR2B Y1472F -V5. Neurons were stimulated with leptin (50 nM) on DIV8, and on DIV11 to 12 spine density was measured by hand using ImageJ with the NeuronJ plugin ( C,D ), or electrophysiological recordings were performed ( E ). White bar = 5 µm. ( D ) Quantification of dendritic spine density from a minimum of 2 to 3 dendritic segments from 15 neurons. ( E ) Quantification of mEPSC frequency, amplitude, and decay time normalized to control condition (control: n = 32; control + leptin: n = 34; NR2B Y1472F : n = 33; NR2B Y1472F + leptin: n = 33). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Journal: Endocrinology

    Article Title: Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B

    doi: 10.1210/endocr/bqz030

    Figure Lengend Snippet: pNR2B Y1472 is necessary for leptin-stimulated spine formation. ( A ) Representative fluorescent images of hippocampal neurons expressing Clover and EGFP-NR2B-V5 or EGFP-NR2B Y1472F -V5 ± leptin stimulation (50 nM, 2 hours) and live immunostained for surface EGFP-NR2B. White bar = 20 µm. ( B ) Quantification of immunostained EGFP-integrated signal density (n = 23). ( C-E ) Hippocampal neurons that were transfected with a fluorescent Clover-βactin and EGFP-NR2B Y1472F -V5. Neurons were stimulated with leptin (50 nM) on DIV8, and on DIV11 to 12 spine density was measured by hand using ImageJ with the NeuronJ plugin ( C,D ), or electrophysiological recordings were performed ( E ). White bar = 5 µm. ( D ) Quantification of dendritic spine density from a minimum of 2 to 3 dendritic segments from 15 neurons. ( E ) Quantification of mEPSC frequency, amplitude, and decay time normalized to control condition (control: n = 32; control + leptin: n = 34; NR2B Y1472F : n = 33; NR2B Y1472F + leptin: n = 33). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Article Snippet: Neurons fixed and incubated with an anti-NR2B (1:100, Alomone Labs) ( ) and anti-Flag (1:250, Sigma Aldrich) ( ) antibody for 1 hour and then incubated with the appropriate Alexa Fluor secondary IgG antibody ( ) for 1 hour at room temperature.

    Techniques: Expressing, Transfection

    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding NR1, NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding NR1, NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    Increased expression of NMDA receptor subunits in renal cortex of Akita mice. A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, and NR2C subunits but not in NR2B or NR2D in renal cortex in 12-week-old Akita mice compared with 12-week-old DBA/2J control mice. B : Densitometric analysis from four mice per group. C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in Akita mice compared with DBA/2J control mice. D : Densitometric analysis from four mice per group. Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Increased expression of NMDA receptor subunits in renal cortex of Akita mice. A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, and NR2C subunits but not in NR2B or NR2D in renal cortex in 12-week-old Akita mice compared with 12-week-old DBA/2J control mice. B : Densitometric analysis from four mice per group. C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in Akita mice compared with DBA/2J control mice. D : Densitometric analysis from four mice per group. Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    Immunohistochemistry (IHC) suggests increased abundance of NMDA receptor subunits throughout the kidney of 12-week-old Akita mice. IHC was carried out in paraffin sections. Negative control sections shown at the top were not exposed to a primary antibody. A : Especially large increases in NR1, NR2A, and NR2C in renal tubules. B : Signal in glomeruli for NR1, NR2A, and NR2C. Primary processes were visible in some of the cells within glomeruli. C : Staining intensity per square micron in the whole kidney (top) and within glomeruli (bottom). Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Immunohistochemistry (IHC) suggests increased abundance of NMDA receptor subunits throughout the kidney of 12-week-old Akita mice. IHC was carried out in paraffin sections. Negative control sections shown at the top were not exposed to a primary antibody. A : Especially large increases in NR1, NR2A, and NR2C in renal tubules. B : Signal in glomeruli for NR1, NR2A, and NR2C. Primary processes were visible in some of the cells within glomeruli. C : Staining intensity per square micron in the whole kidney (top) and within glomeruli (bottom). Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Immunohistochemistry, Mouse Assay, Negative Control, Staining

    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in cultured mouse podocytes (MPC-5 cells). A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, NR2B, and NR2C subunits in cells cultured for 24 h in a medium containing 25 mmol/L glucose (HG). There was no change in NR2D. Control cells (Con) were cultured in medium containing 9 mmol/L glucose, with 16 mmol/L mannitol as an osmotic control. B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in podocytes cultured in HG compared with Con. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Exposure to high glucose (HG) increases expression of NMDA receptor subunits in cultured mouse podocytes (MPC-5 cells). A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, NR2B, and NR2C subunits in cells cultured for 24 h in a medium containing 25 mmol/L glucose (HG). There was no change in NR2D. Control cells (Con) were cultured in medium containing 9 mmol/L glucose, with 16 mmol/L mannitol as an osmotic control. B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in podocytes cultured in HG compared with Con. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Journal: Journal of Radiation Research

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    doi: 10.1093/jrr/rrx032

    Figure Lengend Snippet: Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Article Snippet: Reagents We utilized the following reagents to investigate the effects of chronic, low-dose-rate 137 Cs-γ radiation on NGF-induced neurite extension in PC12 cells: NGF 2.5S (N-100, Alomone Labs, Jerusalem, Israel); anti-ERK1/ERK2 mouse-monoclonal antibody (MAB1576) and anti-phosphorylated ERK1/ERK2 rabbit-polyclonal antibody (MAB1018, both R & D systems, Minneapolis, MN, USA); anti-Trk A rabbit polyclonal antibody (sc-118) and anti-phosphorylated Trk A mouse-monoclonal antibody (sc-8058, both Santa Cruz Biotechnology, Dallas, TX, USA)—Trk A is a high-affinity nerve growth factor receptor; anti-tyrosine hydroxylase rabbit-monoclonal antibody (ab137869); anti-Akt rabbit polyclonal antibody (ab8805) and anti-phosphorylated Akt rabbit monoclonal antibody (ab81283); anti-Ca2+/calmodulin-dependent kinase II (CaMKII) rabbit polyclonal antibody (ab131468) and anti-phosphorylated CaMKII rabbit polyclonal antibody (ab5683, all Abcam plc, Cambridge, UK); KN-62 (I2142, Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Irradiation

    Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.

    Journal: Journal of Radiation Research

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    doi: 10.1093/jrr/rrx032

    Figure Lengend Snippet: Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.

    Article Snippet: Reagents We utilized the following reagents to investigate the effects of chronic, low-dose-rate 137 Cs-γ radiation on NGF-induced neurite extension in PC12 cells: NGF 2.5S (N-100, Alomone Labs, Jerusalem, Israel); anti-ERK1/ERK2 mouse-monoclonal antibody (MAB1576) and anti-phosphorylated ERK1/ERK2 rabbit-polyclonal antibody (MAB1018, both R & D systems, Minneapolis, MN, USA); anti-Trk A rabbit polyclonal antibody (sc-118) and anti-phosphorylated Trk A mouse-monoclonal antibody (sc-8058, both Santa Cruz Biotechnology, Dallas, TX, USA)—Trk A is a high-affinity nerve growth factor receptor; anti-tyrosine hydroxylase rabbit-monoclonal antibody (ab137869); anti-Akt rabbit polyclonal antibody (ab8805) and anti-phosphorylated Akt rabbit monoclonal antibody (ab81283); anti-Ca2+/calmodulin-dependent kinase II (CaMKII) rabbit polyclonal antibody (ab131468) and anti-phosphorylated CaMKII rabbit polyclonal antibody (ab5683, all Abcam plc, Cambridge, UK); KN-62 (I2142, Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Western Blot, Irradiation

    Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Journal: Journal of Radiation Research

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    doi: 10.1093/jrr/rrx032

    Figure Lengend Snippet: Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Article Snippet: Reagents We utilized the following reagents to investigate the effects of chronic, low-dose-rate 137 Cs-γ radiation on NGF-induced neurite extension in PC12 cells: NGF 2.5S (N-100, Alomone Labs, Jerusalem, Israel); anti-ERK1/ERK2 mouse-monoclonal antibody (MAB1576) and anti-phosphorylated ERK1/ERK2 rabbit-polyclonal antibody (MAB1018, both R & D systems, Minneapolis, MN, USA); anti-Trk A rabbit polyclonal antibody (sc-118) and anti-phosphorylated Trk A mouse-monoclonal antibody (sc-8058, both Santa Cruz Biotechnology, Dallas, TX, USA)—Trk A is a high-affinity nerve growth factor receptor; anti-tyrosine hydroxylase rabbit-monoclonal antibody (ab137869); anti-Akt rabbit polyclonal antibody (ab8805) and anti-phosphorylated Akt rabbit monoclonal antibody (ab81283); anti-Ca2+/calmodulin-dependent kinase II (CaMKII) rabbit polyclonal antibody (ab131468) and anti-phosphorylated CaMKII rabbit polyclonal antibody (ab5683, all Abcam plc, Cambridge, UK); KN-62 (I2142, Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Inhibition, Activity Assay, Irradiation

    Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P

    Journal: Journal of Radiation Research

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    doi: 10.1093/jrr/rrx032

    Figure Lengend Snippet: Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P

    Article Snippet: Reagents We utilized the following reagents to investigate the effects of chronic, low-dose-rate 137 Cs-γ radiation on NGF-induced neurite extension in PC12 cells: NGF 2.5S (N-100, Alomone Labs, Jerusalem, Israel); anti-ERK1/ERK2 mouse-monoclonal antibody (MAB1576) and anti-phosphorylated ERK1/ERK2 rabbit-polyclonal antibody (MAB1018, both R & D systems, Minneapolis, MN, USA); anti-Trk A rabbit polyclonal antibody (sc-118) and anti-phosphorylated Trk A mouse-monoclonal antibody (sc-8058, both Santa Cruz Biotechnology, Dallas, TX, USA)—Trk A is a high-affinity nerve growth factor receptor; anti-tyrosine hydroxylase rabbit-monoclonal antibody (ab137869); anti-Akt rabbit polyclonal antibody (ab8805) and anti-phosphorylated Akt rabbit monoclonal antibody (ab81283); anti-Ca2+/calmodulin-dependent kinase II (CaMKII) rabbit polyclonal antibody (ab131468) and anti-phosphorylated CaMKII rabbit polyclonal antibody (ab5683, all Abcam plc, Cambridge, UK); KN-62 (I2142, Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Irradiation, Activation Assay, Activity Assay

    Protein kinase A (PKA)-dependent regulation of endogenous Kv7.5 currents in cultured HASMCs. ( A ) Representative current traces recorded in a single HASMC (Capacitance = 281 pF) before (i. control) and 5 min after addition of 10 µM retigabine (ii). ( B ) Mean fractional conductance plot calculated from steady-state endogenous Kv7 currents fitted to a Boltzmann distribution (V 0.5 = −40.8 mV, n = 10). ( C ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 5), after 5 min treatment with 1 µM forskolin (open circles, n = 4), and after 5 min treatment with diclofenac (100 µM, open triangles, n = 4). ( D ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 7), after 5 min treatment with 1 nM formoterol (open circles, n = 7), and after 5 min treatment with Kv7 channel blocker XE991 (1 µM) in the presence of 1 nM formoterol (closed triangles, n = 3). ( E ) Representative time-courses of 1 nM formoterol application recorded at −20 mV in a single untreated HASMC (black, Capacitance = 39 pF) and a HASMC pretreated with 10 µM H-89 (red, C = 127 pF). ( F ) Relative formoterol-induced enhancement of the current recorded at −20 mV in untreated HASMCs (black bars, n = 7) and in HASMCs pretreated with H-89 (10 µM for 20 min, red bar, n = 4). * Significant difference from control ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Mechanisms of PKA-Dependent Potentiation of Kv7.5 Channel Activity in Human Airway Smooth Muscle Cells

    doi: 10.3390/ijms19082223

    Figure Lengend Snippet: Protein kinase A (PKA)-dependent regulation of endogenous Kv7.5 currents in cultured HASMCs. ( A ) Representative current traces recorded in a single HASMC (Capacitance = 281 pF) before (i. control) and 5 min after addition of 10 µM retigabine (ii). ( B ) Mean fractional conductance plot calculated from steady-state endogenous Kv7 currents fitted to a Boltzmann distribution (V 0.5 = −40.8 mV, n = 10). ( C ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 5), after 5 min treatment with 1 µM forskolin (open circles, n = 4), and after 5 min treatment with diclofenac (100 µM, open triangles, n = 4). ( D ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 7), after 5 min treatment with 1 nM formoterol (open circles, n = 7), and after 5 min treatment with Kv7 channel blocker XE991 (1 µM) in the presence of 1 nM formoterol (closed triangles, n = 3). ( E ) Representative time-courses of 1 nM formoterol application recorded at −20 mV in a single untreated HASMC (black, Capacitance = 39 pF) and a HASMC pretreated with 10 µM H-89 (red, C = 127 pF). ( F ) Relative formoterol-induced enhancement of the current recorded at −20 mV in untreated HASMCs (black bars, n = 7) and in HASMCs pretreated with H-89 (10 µM for 20 min, red bar, n = 4). * Significant difference from control ( p

    Article Snippet: Retigabine was from Alomone Labs (Jerusalem, Israel).

    Techniques: Cell Culture