k252a  (Alomone Labs)


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    Structured Review

    Alomone Labs k252a
    ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM <t>K252a</t> or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P
    K252a, 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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    1) Product Images from "Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway"

    Article Title: Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-15-108

    ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM K252a or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P
    Figure Legend Snippet: ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM K252a or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P

    Techniques Used: Activity Assay, Incubation, Western Blot, Activation Assay

    NGF induces the proteolytic processing of ApoER2. (A) Serum-starved PC12-ApoER2 cells were pre-treated with 10 μM DAPT (γ-secretase complex inhibitor), 50 μM GM6001 (metalloproteases inhibitor) and/or 100 nM K252a (Trk tyrosine kinase activity inhibitor) for 1 h and then incubated with 100 ng/mL NGF for 2 h. The blot shows full-length p75 NTR , p75 NTR CTF, and α-tubulin as a loading control. As described [ 40 ], NGF induced the proteolysis of p75 NTR and, thus, the accumulation of the CTF. This process depends on TrkA tyrosine kinase activity and the metalloproteinases. (B) Cells were pre-treated with 10 μM DAPT for 1 h and then incubated with 100 ng/mL NGF for 2 h. ApoER2 and the proteolytic fragment ApoER2-CTF were recognized using antibodies against the intracellular region of the receptor. α-tubulin is shown as a loading control. (C) Quantification of blot levels of ApoER2-CTF normalized to the loading control α-tubulin and plotted as the average ± SD of four independent experiments. Student’s t-test, ** P
    Figure Legend Snippet: NGF induces the proteolytic processing of ApoER2. (A) Serum-starved PC12-ApoER2 cells were pre-treated with 10 μM DAPT (γ-secretase complex inhibitor), 50 μM GM6001 (metalloproteases inhibitor) and/or 100 nM K252a (Trk tyrosine kinase activity inhibitor) for 1 h and then incubated with 100 ng/mL NGF for 2 h. The blot shows full-length p75 NTR , p75 NTR CTF, and α-tubulin as a loading control. As described [ 40 ], NGF induced the proteolysis of p75 NTR and, thus, the accumulation of the CTF. This process depends on TrkA tyrosine kinase activity and the metalloproteinases. (B) Cells were pre-treated with 10 μM DAPT for 1 h and then incubated with 100 ng/mL NGF for 2 h. ApoER2 and the proteolytic fragment ApoER2-CTF were recognized using antibodies against the intracellular region of the receptor. α-tubulin is shown as a loading control. (C) Quantification of blot levels of ApoER2-CTF normalized to the loading control α-tubulin and plotted as the average ± SD of four independent experiments. Student’s t-test, ** P

    Techniques Used: Activity Assay, Incubation

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    Alomone Labs angiotensin iii
    ANG II mediated increases in sniffer cell fluorescence. ( A ) ANG II (100 nM) induced a robust but transient increase in GCaMP fluorescence that was blocked by the AT1aR receptor antagonist <t>Losartan</t> (10 µM). Control n = 39, Losartan n = 38. ( B ) Data shows that bath application of glutamate (50 µM), GABA (50 µM), and carbachol (50 µM) failed to change fluorescent intensity of sniffer cells transfected with GCaMP (n = 29) or GCaMP + AT1aR (n = 38). ANG II (100 nM) did increase fluorescent intensity of sniffer cells, but only in sniffer cells transfected with GCaMP + AT1aR. Dose-dependent effects of ANG II and related compounds were also measured. ( C ) GCaMP + AT1aR sniffer cells exhibit dose-dependent increases in fluorescence in response to bath application of ANG II (100 nM, n = 10). ( D ) Bath application of ANG <t>III</t> induced a dose-dependent increase in GCaMP + AT1aR sniffer cell fluorescence. Bath application of ANG (1–7) or bradykinin did not induce a change in GCaMP + AT1aR sniffer cell fluorescence at any of the doses tested (0.1–100 nM, n = 17–41). ( E ) R-GECO + AT1aR sniffer cells exhibit dose-dependent increases in fluorescence in response to bath application of ANG II (n = 17). R-GECO only cells did not respond to ANG II (n = 27). ( F ) ANG II-mediated increases in R-GECO + AT1aR are blocked by bath application of Losartan (10 µM, n = 17). *p
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    93
    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
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    93
    Alomone Labs γ2 subunits
    Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 R43Q GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and <t>γ2</t> R43Q subunits with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 R43Q GABA A Rs. The control data for α1β2γ2 and α1β2γ2 R43Q GABA A Rs have been replotted from Figure 1 . All data in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 R43Q GABA A Rs at 40°C. * p
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    95
    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
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    ANG II mediated increases in sniffer cell fluorescence. ( A ) ANG II (100 nM) induced a robust but transient increase in GCaMP fluorescence that was blocked by the AT1aR receptor antagonist Losartan (10 µM). Control n = 39, Losartan n = 38. ( B ) Data shows that bath application of glutamate (50 µM), GABA (50 µM), and carbachol (50 µM) failed to change fluorescent intensity of sniffer cells transfected with GCaMP (n = 29) or GCaMP + AT1aR (n = 38). ANG II (100 nM) did increase fluorescent intensity of sniffer cells, but only in sniffer cells transfected with GCaMP + AT1aR. Dose-dependent effects of ANG II and related compounds were also measured. ( C ) GCaMP + AT1aR sniffer cells exhibit dose-dependent increases in fluorescence in response to bath application of ANG II (100 nM, n = 10). ( D ) Bath application of ANG III induced a dose-dependent increase in GCaMP + AT1aR sniffer cell fluorescence. Bath application of ANG (1–7) or bradykinin did not induce a change in GCaMP + AT1aR sniffer cell fluorescence at any of the doses tested (0.1–100 nM, n = 17–41). ( E ) R-GECO + AT1aR sniffer cells exhibit dose-dependent increases in fluorescence in response to bath application of ANG II (n = 17). R-GECO only cells did not respond to ANG II (n = 27). ( F ) ANG II-mediated increases in R-GECO + AT1aR are blocked by bath application of Losartan (10 µM, n = 17). *p

    Journal: Scientific Reports

    Article Title: Sniffer cells for the detection of neural Angiotensin II in vitro

    doi: 10.1038/s41598-019-45262-4

    Figure Lengend Snippet: ANG II mediated increases in sniffer cell fluorescence. ( A ) ANG II (100 nM) induced a robust but transient increase in GCaMP fluorescence that was blocked by the AT1aR receptor antagonist Losartan (10 µM). Control n = 39, Losartan n = 38. ( B ) Data shows that bath application of glutamate (50 µM), GABA (50 µM), and carbachol (50 µM) failed to change fluorescent intensity of sniffer cells transfected with GCaMP (n = 29) or GCaMP + AT1aR (n = 38). ANG II (100 nM) did increase fluorescent intensity of sniffer cells, but only in sniffer cells transfected with GCaMP + AT1aR. Dose-dependent effects of ANG II and related compounds were also measured. ( C ) GCaMP + AT1aR sniffer cells exhibit dose-dependent increases in fluorescence in response to bath application of ANG II (100 nM, n = 10). ( D ) Bath application of ANG III induced a dose-dependent increase in GCaMP + AT1aR sniffer cell fluorescence. Bath application of ANG (1–7) or bradykinin did not induce a change in GCaMP + AT1aR sniffer cell fluorescence at any of the doses tested (0.1–100 nM, n = 17–41). ( E ) R-GECO + AT1aR sniffer cells exhibit dose-dependent increases in fluorescence in response to bath application of ANG II (n = 17). R-GECO only cells did not respond to ANG II (n = 27). ( F ) ANG II-mediated increases in R-GECO + AT1aR are blocked by bath application of Losartan (10 µM, n = 17). *p

    Article Snippet: Carbachol (50 µM), Angiotensin 1–7 (0.1–100 nM), Bradykinin (0.1–100 nM), and Losartan (10 µM) were purchased from Tocris (Minneapolis, MN) and Angiotensin III (0.1–100 nM) was purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Fluorescence, Transfection

    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

    Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 R43Q GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 R43Q subunits with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 R43Q GABA A Rs. The control data for α1β2γ2 and α1β2γ2 R43Q GABA A Rs have been replotted from Figure 1 . All data in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 R43Q GABA A Rs at 40°C. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

    doi: 10.3389/fnmol.2018.00089

    Figure Lengend Snippet: Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 R43Q GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 R43Q subunits with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 R43Q GABA A Rs. The control data for α1β2γ2 and α1β2γ2 R43Q GABA A Rs have been replotted from Figure 1 . All data in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 R43Q GABA A Rs at 40°C. * p

    Article Snippet: Immunohistochemistry To determine the cell surface expression levels of GABAARs, live HEK293 cells were incubated for 1 h with primary antibodies directed against surface epitopes of γ2 subunits (1:100, rabbit, #GA-005, Alomone Labs, Jerusalem, Israel) in equilibrated DMEM containing 1% BSA at 37°C.

    Techniques: Transfection

    Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 K289M GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 K289M subunits with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 K289M GABA A Rs. The control data for α1β2γ2 and α1β2γ2 K289M GABA A Rs have been replotted from Figure 1 . All results in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 K289M GABA A Rs at 40°C. ** p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

    doi: 10.3389/fnmol.2018.00089

    Figure Lengend Snippet: Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 K289M GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 K289M subunits with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 K289M GABA A Rs. The control data for α1β2γ2 and α1β2γ2 K289M GABA A Rs have been replotted from Figure 1 . All results in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 K289M GABA A Rs at 40°C. ** p

    Article Snippet: Immunohistochemistry To determine the cell surface expression levels of GABAARs, live HEK293 cells were incubated for 1 h with primary antibodies directed against surface epitopes of γ2 subunits (1:100, rabbit, #GA-005, Alomone Labs, Jerusalem, Israel) in equilibrated DMEM containing 1% BSA at 37°C.

    Techniques: Transfection

    Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 R138G GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 R138G subunits s with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 R138G GABA A Rs. The control data for α1β2γ2 and α1β2γ2 R138G GABA A Rs have been replotted from Figure 1 . All results in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10% –90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 R138G GABA A Rs at 40°C. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

    doi: 10.3389/fnmol.2018.00089

    Figure Lengend Snippet: Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 R138G GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 R138G subunits s with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 R138G GABA A Rs. The control data for α1β2γ2 and α1β2γ2 R138G GABA A Rs have been replotted from Figure 1 . All results in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10% –90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 R138G GABA A Rs at 40°C. * p

    Article Snippet: Immunohistochemistry To determine the cell surface expression levels of GABAARs, live HEK293 cells were incubated for 1 h with primary antibodies directed against surface epitopes of γ2 subunits (1:100, rabbit, #GA-005, Alomone Labs, Jerusalem, Israel) in equilibrated DMEM containing 1% BSA at 37°C.

    Techniques: Transfection

    Effect of suberanilohydroxamic acid (SAHA) on the surface expression of wild type and mutant GABA A Rs. (A) Sample image of HEK293 cells expressing α1β2γ2 GABA A Rs. The scale bar represents 10 μm. (B) Sample image of HEK293 cells expressing α1β2γ2 R43Q GABA A Rs. (C) Sample image of HEK293 cells expressing α1β2γ2 R43Q GABA A Rs following SAHA pre-application. (D) In the absence of SAHA pre-application, all mutants except γ2 K298M showed reduced surface expression relative to wild type (20–60 cells per mutant). (E) After SAHA pre-application, the surface expression levels of all mutants were not significantly different to wild type. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

    doi: 10.3389/fnmol.2018.00089

    Figure Lengend Snippet: Effect of suberanilohydroxamic acid (SAHA) on the surface expression of wild type and mutant GABA A Rs. (A) Sample image of HEK293 cells expressing α1β2γ2 GABA A Rs. The scale bar represents 10 μm. (B) Sample image of HEK293 cells expressing α1β2γ2 R43Q GABA A Rs. (C) Sample image of HEK293 cells expressing α1β2γ2 R43Q GABA A Rs following SAHA pre-application. (D) In the absence of SAHA pre-application, all mutants except γ2 K298M showed reduced surface expression relative to wild type (20–60 cells per mutant). (E) After SAHA pre-application, the surface expression levels of all mutants were not significantly different to wild type. * p

    Article Snippet: Immunohistochemistry To determine the cell surface expression levels of GABAARs, live HEK293 cells were incubated for 1 h with primary antibodies directed against surface epitopes of γ2 subunits (1:100, rabbit, #GA-005, Alomone Labs, Jerusalem, Israel) in equilibrated DMEM containing 1% BSA at 37°C.

    Techniques: Expressing, Mutagenesis

    Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 N40S GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 N40S subunits with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 N40S GABA A Rs. The control data for α1β2γ2 and α1β2γ2 N40S GABA A Rs have been replotted from Figure 1 . All data in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 N40S GABA A Rs at 40°C. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

    doi: 10.3389/fnmol.2018.00089

    Figure Lengend Snippet: Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 N40S GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 N40S subunits with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 N40S GABA A Rs. The control data for α1β2γ2 and α1β2γ2 N40S GABA A Rs have been replotted from Figure 1 . All data in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 N40S GABA A Rs at 40°C. * p

    Article Snippet: Immunohistochemistry To determine the cell surface expression levels of GABAARs, live HEK293 cells were incubated for 1 h with primary antibodies directed against surface epitopes of γ2 subunits (1:100, rabbit, #GA-005, Alomone Labs, Jerusalem, Israel) in equilibrated DMEM containing 1% BSA at 37°C.

    Techniques: Transfection

    Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 P44S GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 P44S subunits with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 P44S GABA A Rs. The control data for α1β2γ2 and α1β2γ2 P44S GABA A Rs have been replotted from Figure 1 . All results in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 P44S GABA A Rs at 40°C. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

    doi: 10.3389/fnmol.2018.00089

    Figure Lengend Snippet: Effects of SAHA and temperature on IPSCs mediated by α1β2γ2 P44S GABA A Rs. (A) Examples of digitally averaged and normalised IPSCs from HEK293 cells transfected with α1, β2 and γ2 P44S subunits with and without SAHA pre-application. (B–D) Effect of SAHA pre-application on the amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 P44S GABA A Rs. The control data for α1β2γ2 and α1β2γ2 P44S GABA A Rs have been replotted from Figure 1 . All results in panels (A–D) were recorded at room temperature (22°C). (E–G) Mean amplitudes, 10%–90% rise times and decay time constants of IPSCs mediated by α1β2γ2 and α1β2γ2 P44S GABA A Rs at 40°C. * p

    Article Snippet: Immunohistochemistry To determine the cell surface expression levels of GABAARs, live HEK293 cells were incubated for 1 h with primary antibodies directed against surface epitopes of γ2 subunits (1:100, rabbit, #GA-005, Alomone Labs, Jerusalem, Israel) in equilibrated DMEM containing 1% BSA at 37°C.

    Techniques: 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