guinea pig anti nmdar1 glun1 extracellular antibody  (Alomone Labs)


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    Alomone Labs guinea pig anti nmdar1 glun1 extracellular antibody
    Inhibiting GluN2C NMDARs reduces PPR variability in a manner dependent on astrocyte expression of <t>GluN1.</t> ( A–C ) Left: representative EPSC traces (average of 20 sweeps) to pairs of pulses (Δt = 50 ms) applied to Schaffer collateral axons in baseline and after wash-on of QNZ46 (25 µM). Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) before and after bath applying QNZ46 to control uninfected ( A ), Control ( B ) and Cre ( C ) slices. The p values were obtained for the population mean ( x - ) (paired sample t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients ( r ) and p values are shown in scatter plots. ( D–F ) Plots of EPSC amplitudes (normalized to the baseline average) before and during the application of QNZ46 to control uninfected ( D ), Control ( E ) and Cre-infected ( F ) slices (shaded area) where n is the number of inputs examined for each experiment. Baseline and experimental periods are indicated (black bars). Box plots show the summary of normalized EPSC amplitudes during the QNZ46 application. *p
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    1) Product Images from "Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum"

    Article Title: Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum

    Journal: eLife

    doi: 10.7554/eLife.70818

    Inhibiting GluN2C NMDARs reduces PPR variability in a manner dependent on astrocyte expression of GluN1. ( A–C ) Left: representative EPSC traces (average of 20 sweeps) to pairs of pulses (Δt = 50 ms) applied to Schaffer collateral axons in baseline and after wash-on of QNZ46 (25 µM). Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) before and after bath applying QNZ46 to control uninfected ( A ), Control ( B ) and Cre ( C ) slices. The p values were obtained for the population mean ( x - ) (paired sample t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients ( r ) and p values are shown in scatter plots. ( D–F ) Plots of EPSC amplitudes (normalized to the baseline average) before and during the application of QNZ46 to control uninfected ( D ), Control ( E ) and Cre-infected ( F ) slices (shaded area) where n is the number of inputs examined for each experiment. Baseline and experimental periods are indicated (black bars). Box plots show the summary of normalized EPSC amplitudes during the QNZ46 application. *p
    Figure Legend Snippet: Inhibiting GluN2C NMDARs reduces PPR variability in a manner dependent on astrocyte expression of GluN1. ( A–C ) Left: representative EPSC traces (average of 20 sweeps) to pairs of pulses (Δt = 50 ms) applied to Schaffer collateral axons in baseline and after wash-on of QNZ46 (25 µM). Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) before and after bath applying QNZ46 to control uninfected ( A ), Control ( B ) and Cre ( C ) slices. The p values were obtained for the population mean ( x - ) (paired sample t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients ( r ) and p values are shown in scatter plots. ( D–F ) Plots of EPSC amplitudes (normalized to the baseline average) before and during the application of QNZ46 to control uninfected ( D ), Control ( E ) and Cre-infected ( F ) slices (shaded area) where n is the number of inputs examined for each experiment. Baseline and experimental periods are indicated (black bars). Box plots show the summary of normalized EPSC amplitudes during the QNZ46 application. *p

    Techniques Used: Expressing, One-tailed Test, Infection

    Knock-down of GluN1 in hippocampal astrocytes selectively alters the PPR disparity without affecting the basic properties of synaptic transmission. [Related also to Figure 6 ] ( A ) Left: Illustration of the experimental configuration of two-pathway stimulation approach to estimate PPR disparity in slices from Grin1 flx/flx mice infected with Control or Cre virus. Right: Comparison of ΔPPR (|PPR S1 -PPR S2 |) between Control or Cre slices. PPR disparity (ΔPPR) is reduced in Cre infected slices compared to controls. p = 0.009, Mann-Whitney U test. Control n = 29 pairs of inputs, 29 cells, 8 mice. Cre n = 28 pairs of inputs, 28 cells, 6 mice. ( B ) Left: Grand average waveforms of EPSCs recorded. Right: Box plots of EPSC risetime and EPSC decay time constant (single exponential fit). Each data point represents the average waveform of at least 5 sweeps of EPSCs. Control n = 46 inputs, 23 cells, 8 mice. Cre n = 41 inputs, 21 cells, 6 mice. ( C ) Left: Representative traces of spontaneous EPSC (sEPSC) recordings from Control (blue) or Cre (red) slices. Right: Boxplots of sEPSC amplitude and frequency measured in Control or Cre-infected slices. P values shown are the result of Mann-Whitney U test. Control n = 23 cells, 5 mice. Cre n = 26 cells, 5 mice. ( D–G ) Normalized sEPSC amplitude (top) and frequency (bottom) during baseline and in the presence of AP5 or QNZ46 in Control or Cre-infected slices. Neither sEPSC amplitude nor frequency is not affected by AP5 or QNZ46. Two tailed t-test or Mann-Whitney U test, comparisons made between Control vs. Cre normalized values in post drug period indicated. ( H–K ) ΔPPR (|PPR S1 -PPR S2 |) measurements during baseline and in the presence of AP5 or QNZ46 in Control or Cre-infected slices. The decrease in PPR disparity triggered by AP5 ( H ) and QNZ46 ( J ) is prevented upon knock-down of astrocyte NMDARs. * p
    Figure Legend Snippet: Knock-down of GluN1 in hippocampal astrocytes selectively alters the PPR disparity without affecting the basic properties of synaptic transmission. [Related also to Figure 6 ] ( A ) Left: Illustration of the experimental configuration of two-pathway stimulation approach to estimate PPR disparity in slices from Grin1 flx/flx mice infected with Control or Cre virus. Right: Comparison of ΔPPR (|PPR S1 -PPR S2 |) between Control or Cre slices. PPR disparity (ΔPPR) is reduced in Cre infected slices compared to controls. p = 0.009, Mann-Whitney U test. Control n = 29 pairs of inputs, 29 cells, 8 mice. Cre n = 28 pairs of inputs, 28 cells, 6 mice. ( B ) Left: Grand average waveforms of EPSCs recorded. Right: Box plots of EPSC risetime and EPSC decay time constant (single exponential fit). Each data point represents the average waveform of at least 5 sweeps of EPSCs. Control n = 46 inputs, 23 cells, 8 mice. Cre n = 41 inputs, 21 cells, 6 mice. ( C ) Left: Representative traces of spontaneous EPSC (sEPSC) recordings from Control (blue) or Cre (red) slices. Right: Boxplots of sEPSC amplitude and frequency measured in Control or Cre-infected slices. P values shown are the result of Mann-Whitney U test. Control n = 23 cells, 5 mice. Cre n = 26 cells, 5 mice. ( D–G ) Normalized sEPSC amplitude (top) and frequency (bottom) during baseline and in the presence of AP5 or QNZ46 in Control or Cre-infected slices. Neither sEPSC amplitude nor frequency is not affected by AP5 or QNZ46. Two tailed t-test or Mann-Whitney U test, comparisons made between Control vs. Cre normalized values in post drug period indicated. ( H–K ) ΔPPR (|PPR S1 -PPR S2 |) measurements during baseline and in the presence of AP5 or QNZ46 in Control or Cre-infected slices. The decrease in PPR disparity triggered by AP5 ( H ) and QNZ46 ( J ) is prevented upon knock-down of astrocyte NMDARs. * p

    Techniques Used: Transmission Assay, Mouse Assay, Infection, MANN-WHITNEY, Two Tailed Test

    Adult mouse hippocampal astrocytes express GluN2C-NMDA receptors. ( A ) Top: patch RT-PCR strategy. Bottom: representative voltage responses to current steps in CA1 pyramidal neuron (far left, black), and current responses to voltage steps in astrocytes in stratum radiatum (SR, green), stratum oriens (SO, yellow), and stratum lacunosum moleculare (SLM, blue). Scale bars: neuron, 40 mV, 500 ms; astrocytes, 6 nA, 200 ms. ( B ) Summary of I-V curves of astrocytes obtained before collecting RNA. N = 39 cells, 36 slices, 9 mice for each layer. ( C ) Summary of RT-PCR analysis for mRNA encoding indicated NMDAR subunits: Grin1 (1), Grin2a (2A), Grin2b (2B), and Grin2c (2C). Control samples (no patch) were obtained by inserting the electrode into the slice without patching cells. Three technical replicates were performed for each condition. ( D ) Western blots of co-immunoprecipitations performed using adult mouse hippocampal extracts with the indicated antibodies where anti-Cre antibody is used as a negative control. The blots are probed for GluN2A/2B (top row), GluN2C (middle row) or GluN1 (bottom row). ( E ) Representative brain sections of Grin2c iCre/+ mice crossed to a tdTomato reporter line (Ai9), showing hippocampal area CA1 immunolabelled for GFAP, tdTomato and NeuN; scale bars, 160 μm (top), 25 μm (bottom). ( F ) Quantification of % cells that are positive for tdTomato amongst the counts of NeuN-labelled cells in stratum pyramidale (left, total n = 3155 cells) and SR (middle, total n = 619 cells), and GFAP-labelled cells (right, total n = 3149 cells). N = 10 fields of view of area CA1 from individual hippocampal sections (n = 10) obtained from 5 mice. Data are presented as mean ± s.e.m.
    Figure Legend Snippet: Adult mouse hippocampal astrocytes express GluN2C-NMDA receptors. ( A ) Top: patch RT-PCR strategy. Bottom: representative voltage responses to current steps in CA1 pyramidal neuron (far left, black), and current responses to voltage steps in astrocytes in stratum radiatum (SR, green), stratum oriens (SO, yellow), and stratum lacunosum moleculare (SLM, blue). Scale bars: neuron, 40 mV, 500 ms; astrocytes, 6 nA, 200 ms. ( B ) Summary of I-V curves of astrocytes obtained before collecting RNA. N = 39 cells, 36 slices, 9 mice for each layer. ( C ) Summary of RT-PCR analysis for mRNA encoding indicated NMDAR subunits: Grin1 (1), Grin2a (2A), Grin2b (2B), and Grin2c (2C). Control samples (no patch) were obtained by inserting the electrode into the slice without patching cells. Three technical replicates were performed for each condition. ( D ) Western blots of co-immunoprecipitations performed using adult mouse hippocampal extracts with the indicated antibodies where anti-Cre antibody is used as a negative control. The blots are probed for GluN2A/2B (top row), GluN2C (middle row) or GluN1 (bottom row). ( E ) Representative brain sections of Grin2c iCre/+ mice crossed to a tdTomato reporter line (Ai9), showing hippocampal area CA1 immunolabelled for GFAP, tdTomato and NeuN; scale bars, 160 μm (top), 25 μm (bottom). ( F ) Quantification of % cells that are positive for tdTomato amongst the counts of NeuN-labelled cells in stratum pyramidale (left, total n = 3155 cells) and SR (middle, total n = 619 cells), and GFAP-labelled cells (right, total n = 3149 cells). N = 10 fields of view of area CA1 from individual hippocampal sections (n = 10) obtained from 5 mice. Data are presented as mean ± s.e.m.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Western Blot, Negative Control

    2) Product Images from "Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum"

    Article Title: Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum

    Journal: bioRxiv

    doi: 10.1101/2021.05.28.446253

    Knock-down of GluN1 in hippocampal astrocytes selectively alters the PPR disparity without affecting the basic properties of synaptic transmission. ( A ) Left: Illustration of the experimental configuration of two-pathway stimulation approach to estimate PPR disparity in slices from GRIN1 flx/flx mice infected with ΔCre or Cre virus. Right: Comparison of ΔPPR (|PPR S1 -PPR S2 |) between control ΔCre or Cre slices. PPR disparity (ΔPPR) is reduced in Cre infected slices compared to controls. p = .009, Mann-Whitney U test. ΔCre n = 29 pairs of inputs, 29 cells, 8 mice. Cre n = 28 pairs of inputs, 28 cells, 6 mice. ( B ) Left: Grand average waveforms of EPSCs recorded. Right: Box plots of EPSC risetime and EPSC decay time constant (single exponential fit). Each data point represents the average waveform of at least 5 sweeps of EPSCs. ΔCre n = 46 inputs, 23 cells, 8 mice. Cre n = 41 inputs, 21 cells, 6 mice. ( C ) Left: Representative traces of spontaneous EPSC (sEPSC) recordings from control ΔCre (blue) or Cre (red) slices. Right: Boxplots of sEPSC amplitude and frequency measured in ΔCre or Cre-infected slices. P values shown are the result of Mann-Whitney U test. ΔCre n = 23 cells, 5 mice. Cre n = 26 cells, 5 mice. ( D-G ) Normalized sEPSC amplitude (top) and frequency (bottom) during baseline and in the presence of AP5 or QNZ46 in control ΔCre or Cre-infected slices. Neither sEPSC amplitude nor frequency is not affected by AP5 or QNZ46. Two tailed t-test or Mann-Whitney U test, comparisons made between ΔCre vs. Cre normalized values in post drug period indicated. ( H-K ) ΔPPR (|PPR S1 -PPR S2 |) measurements during baseline and in the presence of AP5 or QNZ46 in control ΔCre or Cre-infected slices. The decrease in PPR disparity triggered by AP5 ( H ) and QNZ46 ( J ) is prevented upon knock-down of astrocyte NMDARs. * p
    Figure Legend Snippet: Knock-down of GluN1 in hippocampal astrocytes selectively alters the PPR disparity without affecting the basic properties of synaptic transmission. ( A ) Left: Illustration of the experimental configuration of two-pathway stimulation approach to estimate PPR disparity in slices from GRIN1 flx/flx mice infected with ΔCre or Cre virus. Right: Comparison of ΔPPR (|PPR S1 -PPR S2 |) between control ΔCre or Cre slices. PPR disparity (ΔPPR) is reduced in Cre infected slices compared to controls. p = .009, Mann-Whitney U test. ΔCre n = 29 pairs of inputs, 29 cells, 8 mice. Cre n = 28 pairs of inputs, 28 cells, 6 mice. ( B ) Left: Grand average waveforms of EPSCs recorded. Right: Box plots of EPSC risetime and EPSC decay time constant (single exponential fit). Each data point represents the average waveform of at least 5 sweeps of EPSCs. ΔCre n = 46 inputs, 23 cells, 8 mice. Cre n = 41 inputs, 21 cells, 6 mice. ( C ) Left: Representative traces of spontaneous EPSC (sEPSC) recordings from control ΔCre (blue) or Cre (red) slices. Right: Boxplots of sEPSC amplitude and frequency measured in ΔCre or Cre-infected slices. P values shown are the result of Mann-Whitney U test. ΔCre n = 23 cells, 5 mice. Cre n = 26 cells, 5 mice. ( D-G ) Normalized sEPSC amplitude (top) and frequency (bottom) during baseline and in the presence of AP5 or QNZ46 in control ΔCre or Cre-infected slices. Neither sEPSC amplitude nor frequency is not affected by AP5 or QNZ46. Two tailed t-test or Mann-Whitney U test, comparisons made between ΔCre vs. Cre normalized values in post drug period indicated. ( H-K ) ΔPPR (|PPR S1 -PPR S2 |) measurements during baseline and in the presence of AP5 or QNZ46 in control ΔCre or Cre-infected slices. The decrease in PPR disparity triggered by AP5 ( H ) and QNZ46 ( J ) is prevented upon knock-down of astrocyte NMDARs. * p

    Techniques Used: Transmission Assay, Mouse Assay, Infection, MANN-WHITNEY, Two Tailed Test

    Astrocyte NMDARs mediate the antagonist-induced decrease in synaptic strength diversity. ( A ) Astrocyte GluN1 was conditionally knocked down by injecting AAV-GFAP104-nls-mCherry-Cre (Cre), or as a control, AAV-GFAP104-nls-mCherry (ΔCre), to the dorsal hippocampus of adult GRIN1 flx/flx mice. ( B ) Representative image of hippocampal area CA1 immunolabelled for GFAP (green) and mCherry (red) and counterstained with DAPI (blue), 21 days after virus injection. SO, stratum oriens ; SPy, stratum pyramidale ; SR, stratum radiatum ; SLM, stratum lacunosum moleculare ; MOL, molecular layer; GCL, granule cell layer. Right: magnified view in SR showing restricted expression of mCherry to GFAP-positive astrocytes (arrowheads). Scale bar, 20 µm. ( C ) Recording scheme: CA1 pyramidal neurons were patched in acute slices prepared 21 days after ΔCre or Cre AAV infection. EPSCs were elicited by placing the stimulating electrode in the SR. Patch pipette contained MK801 for experiments in E-H . ( D ) Left: Averaged EPSC traces from representative recordings in slices from control ΔCre (top) or Cre (bottom) virus-infected mice. Individual traces are shown in grey. Right: Histograms of PPRs recorded from ΔCre (top) and Cre slices (bottom) were fit with single Gaussians. The p values were obtained for the population mean (x̅) (two-tailed t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPR distributions from ΔCre (n=72 inputs, 43 cells, 14 mice) and Cre (n=74 inputs, 46 cells, 12 mice) slices. ( E,F ) Left: average EPSC traces in baseline (grey) and after drug treatment (color) from a representative experiment. Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) in baseline and after bath applying AP5 (50 µM) to ΔCre ( E ) and Cre ( F ) slices. The p values for the population mean (x̅) and variance (δ 2 ) are as in D , comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients (r) and p values are shown in scatter plots. ( G,H ) Plots of normalized EPSC amplitudes before and during the application of AP5 to ΔCre ( G ) and Cre-infected ( H ) slices (shaded area); n is the number of inputs examined. Right: summary bar graph. **p = 0.005, Mann-Whitney U-test. ΔCre +AP5 = 22 inputs, 12 cells, 5 mice; Cre +AP5 = 24 inputs, 14 cells, 5 mice. ( Figure 2 legend continued from previous page )
    Figure Legend Snippet: Astrocyte NMDARs mediate the antagonist-induced decrease in synaptic strength diversity. ( A ) Astrocyte GluN1 was conditionally knocked down by injecting AAV-GFAP104-nls-mCherry-Cre (Cre), or as a control, AAV-GFAP104-nls-mCherry (ΔCre), to the dorsal hippocampus of adult GRIN1 flx/flx mice. ( B ) Representative image of hippocampal area CA1 immunolabelled for GFAP (green) and mCherry (red) and counterstained with DAPI (blue), 21 days after virus injection. SO, stratum oriens ; SPy, stratum pyramidale ; SR, stratum radiatum ; SLM, stratum lacunosum moleculare ; MOL, molecular layer; GCL, granule cell layer. Right: magnified view in SR showing restricted expression of mCherry to GFAP-positive astrocytes (arrowheads). Scale bar, 20 µm. ( C ) Recording scheme: CA1 pyramidal neurons were patched in acute slices prepared 21 days after ΔCre or Cre AAV infection. EPSCs were elicited by placing the stimulating electrode in the SR. Patch pipette contained MK801 for experiments in E-H . ( D ) Left: Averaged EPSC traces from representative recordings in slices from control ΔCre (top) or Cre (bottom) virus-infected mice. Individual traces are shown in grey. Right: Histograms of PPRs recorded from ΔCre (top) and Cre slices (bottom) were fit with single Gaussians. The p values were obtained for the population mean (x̅) (two-tailed t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPR distributions from ΔCre (n=72 inputs, 43 cells, 14 mice) and Cre (n=74 inputs, 46 cells, 12 mice) slices. ( E,F ) Left: average EPSC traces in baseline (grey) and after drug treatment (color) from a representative experiment. Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) in baseline and after bath applying AP5 (50 µM) to ΔCre ( E ) and Cre ( F ) slices. The p values for the population mean (x̅) and variance (δ 2 ) are as in D , comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients (r) and p values are shown in scatter plots. ( G,H ) Plots of normalized EPSC amplitudes before and during the application of AP5 to ΔCre ( G ) and Cre-infected ( H ) slices (shaded area); n is the number of inputs examined. Right: summary bar graph. **p = 0.005, Mann-Whitney U-test. ΔCre +AP5 = 22 inputs, 12 cells, 5 mice; Cre +AP5 = 24 inputs, 14 cells, 5 mice. ( Figure 2 legend continued from previous page )

    Techniques Used: Mouse Assay, Injection, Expressing, Infection, Transferring, Two Tailed Test, One-tailed Test, MANN-WHITNEY, Polyacrylamide Gel Electrophoresis

    Puff application of NMDA and glycine triggers the depolarization of astrocytes in hippocampal CA1 in acute slices. ( A ) Left: experimental setup. Middle: Local puff application (100 ms, black bar) of NMDA and glycine (1mM each) to astrocytes that are whole-cell patch clamped in the stratum radiatum (SR) elicits slow membrane depolarization (ΔCre, blue), which is prevented in the presence of AP5 (arrow). Once the depolarization is triggered, not only NMDARs but also other voltage-gated channels such as L-type calcium channels contribute to the slow depolarization ( Letellier et al., 2016 ). The depolarization is substantially reduced when the same experiment is carried from Cre-expressing astrocytes as monitored by mCherry expression in slices infected with the AAV-GFAP104-nls-mCherry-Cre virus to knock-down astrocyte NMDARs in GRIN1 floxed mice (Cre, red). Right: Summary plot of the peak membrane depolarization in control virus infected (ΔCre) or NMDAR knock-down (Cre) slices in control aCSF or the presence of AP5. ( B-C ) Same as ( A ) except NMDA and glycine puff was applied to astrocytes patch-clamped in the stratum oriens (SO)( B ) and stratum lacunosum moleculare (SLM)( C ). NMDA/glycine puff-mediated depolarizations elicited in SO and SLM astrocytes are not sensitive to astrocyte NMDAR knockout. SR ΔCre n = 14 cells, 3 mice; SR Cre n = 14 cells, 3 mice. SO ΔCre n = 8 cells, 3 mice; SO Cre n = 8 cells, 3 mice. SLM ΔCre n = 9 cells, 2 mice; SLM Cre n = 8 cells, 2 mice. ( D ) Cre expression does not alter the input resistance (R in ) of astrocytes in any layer. IV relationships of ΔCre and Cre positive astrocytes in the SR, SO, and SLM to square voltage steps (left; 500ms duration) and quantification of R in (right). SR ΔCre n = 17 cells, 8 mice; SR Cre n = 20 cells, 10 mice. SO ΔCre n = 16 cells, 10 mice; SO Cre n = 16 cells, 11 mice. SLM ΔCre n = 19 cells, 8 mice; SLM Cre n = 18 cells, 9 mice. ns p > .05, P values shown are the result of two-tailed t-tests or Mann-Whitney U tests.
    Figure Legend Snippet: Puff application of NMDA and glycine triggers the depolarization of astrocytes in hippocampal CA1 in acute slices. ( A ) Left: experimental setup. Middle: Local puff application (100 ms, black bar) of NMDA and glycine (1mM each) to astrocytes that are whole-cell patch clamped in the stratum radiatum (SR) elicits slow membrane depolarization (ΔCre, blue), which is prevented in the presence of AP5 (arrow). Once the depolarization is triggered, not only NMDARs but also other voltage-gated channels such as L-type calcium channels contribute to the slow depolarization ( Letellier et al., 2016 ). The depolarization is substantially reduced when the same experiment is carried from Cre-expressing astrocytes as monitored by mCherry expression in slices infected with the AAV-GFAP104-nls-mCherry-Cre virus to knock-down astrocyte NMDARs in GRIN1 floxed mice (Cre, red). Right: Summary plot of the peak membrane depolarization in control virus infected (ΔCre) or NMDAR knock-down (Cre) slices in control aCSF or the presence of AP5. ( B-C ) Same as ( A ) except NMDA and glycine puff was applied to astrocytes patch-clamped in the stratum oriens (SO)( B ) and stratum lacunosum moleculare (SLM)( C ). NMDA/glycine puff-mediated depolarizations elicited in SO and SLM astrocytes are not sensitive to astrocyte NMDAR knockout. SR ΔCre n = 14 cells, 3 mice; SR Cre n = 14 cells, 3 mice. SO ΔCre n = 8 cells, 3 mice; SO Cre n = 8 cells, 3 mice. SLM ΔCre n = 9 cells, 2 mice; SLM Cre n = 8 cells, 2 mice. ( D ) Cre expression does not alter the input resistance (R in ) of astrocytes in any layer. IV relationships of ΔCre and Cre positive astrocytes in the SR, SO, and SLM to square voltage steps (left; 500ms duration) and quantification of R in (right). SR ΔCre n = 17 cells, 8 mice; SR Cre n = 20 cells, 10 mice. SO ΔCre n = 16 cells, 10 mice; SO Cre n = 16 cells, 11 mice. SLM ΔCre n = 19 cells, 8 mice; SLM Cre n = 18 cells, 9 mice. ns p > .05, P values shown are the result of two-tailed t-tests or Mann-Whitney U tests.

    Techniques Used: Expressing, Infection, Mouse Assay, Knock-Out, Two Tailed Test, MANN-WHITNEY

    Adult mouse hippocampal astrocytes express GluN2C-NMDA receptors. ( A ) Top: patch RT-PCR strategy. Bottom: representative voltage responses to current steps in CA1 pyramidal neuron (far left, black), and current responses to voltage steps in astrocytes in stratum radiatum (SR, green), stratum oriens (SO, yellow), and stratum lacunosum moleculare (SLM, blue). Scale bars: neuron, 40 mV, 500 ms; astrocytes, 6 nA, 200 ms. ( B ) Summary of I-V curves of astrocytes obtained before collecting RNA. N = 39 cells, 36 slices, 9 mice for each layer. ( C ) Summary of RT-PCR analysis for mRNA encoding indicated NMDAR subunits: GRIN1 (1), GRIN2A (2A), GRIN2B (2B), and GRIN2C (2C). Control samples (no patch) were obtained by inserting the electrode into the slice without patching cells. GRIN2D levels were undetectable in all cells examined (data not shown). ( D ) Western blots of co-immunoprecipitations performed using adult mouse hippocampal extracts with the indicated antibodies where anti-Cre antibody is used as a negative control. The blots are probed for GluN2A/2B (top row), GluN2C (middle row) or GluN1 (bottom row). ( E ) Representative brain sections of GRIN2C -Cre mice crossed to a tdTomato reporter line (Ai9), showing hippocampal area CA1 immunolabelled for GFAP, tdTomato and NeuN; scale bars, 160 μm (top), 25 μm (bottom). ( F ) Quantification of % cells that are positive for tdTomato amongst NeuN-labelled cells in stratum pyramidale (left) and SR (middle) and GFAP-labelled cells (right).
    Figure Legend Snippet: Adult mouse hippocampal astrocytes express GluN2C-NMDA receptors. ( A ) Top: patch RT-PCR strategy. Bottom: representative voltage responses to current steps in CA1 pyramidal neuron (far left, black), and current responses to voltage steps in astrocytes in stratum radiatum (SR, green), stratum oriens (SO, yellow), and stratum lacunosum moleculare (SLM, blue). Scale bars: neuron, 40 mV, 500 ms; astrocytes, 6 nA, 200 ms. ( B ) Summary of I-V curves of astrocytes obtained before collecting RNA. N = 39 cells, 36 slices, 9 mice for each layer. ( C ) Summary of RT-PCR analysis for mRNA encoding indicated NMDAR subunits: GRIN1 (1), GRIN2A (2A), GRIN2B (2B), and GRIN2C (2C). Control samples (no patch) were obtained by inserting the electrode into the slice without patching cells. GRIN2D levels were undetectable in all cells examined (data not shown). ( D ) Western blots of co-immunoprecipitations performed using adult mouse hippocampal extracts with the indicated antibodies where anti-Cre antibody is used as a negative control. The blots are probed for GluN2A/2B (top row), GluN2C (middle row) or GluN1 (bottom row). ( E ) Representative brain sections of GRIN2C -Cre mice crossed to a tdTomato reporter line (Ai9), showing hippocampal area CA1 immunolabelled for GFAP, tdTomato and NeuN; scale bars, 160 μm (top), 25 μm (bottom). ( F ) Quantification of % cells that are positive for tdTomato amongst NeuN-labelled cells in stratum pyramidale (left) and SR (middle) and GFAP-labelled cells (right).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Western Blot, Negative Control

    Inhibiting GluN2C NMDARs reduces PPR variability in a manner dependent on astrocyte expression of GluN1. ( A-C ) Left: representative EPSC traces (average of 20 sweeps) to pairs of pulses (Δt = 50 ms) applied to Schaffer collateral axons in baseline and after wash-on of QNZ46 (25 µM). Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) before and after bath applying QNZ46 to control uninfected ( A ), ΔCre ( B ) and Cre ( C ) slices. The p values were obtained for the population mean (x̅) (paired sample t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients (r) and p values are shown in scatter plots. ( D-F ) Plots of EPSC amplitudes (normalized to the baseline average) before and during the application of QNZ46 to control uninfected ( D ), ΔCre ( E ) and Cre-infected ( F ) slices (shaded area) where n is the number of inputs examined for each experiment. Baseline and experimental periods are indicated (black bars). Box plots show the summary of normalized EPSC amplitudes during the QNZ46 application. *p
    Figure Legend Snippet: Inhibiting GluN2C NMDARs reduces PPR variability in a manner dependent on astrocyte expression of GluN1. ( A-C ) Left: representative EPSC traces (average of 20 sweeps) to pairs of pulses (Δt = 50 ms) applied to Schaffer collateral axons in baseline and after wash-on of QNZ46 (25 µM). Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) before and after bath applying QNZ46 to control uninfected ( A ), ΔCre ( B ) and Cre ( C ) slices. The p values were obtained for the population mean (x̅) (paired sample t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients (r) and p values are shown in scatter plots. ( D-F ) Plots of EPSC amplitudes (normalized to the baseline average) before and during the application of QNZ46 to control uninfected ( D ), ΔCre ( E ) and Cre-infected ( F ) slices (shaded area) where n is the number of inputs examined for each experiment. Baseline and experimental periods are indicated (black bars). Box plots show the summary of normalized EPSC amplitudes during the QNZ46 application. *p

    Techniques Used: Expressing, One-tailed Test, Infection

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    Alomone Labs guinea pig anti nmdar1 glun1 extracellular antibody
    Inhibiting GluN2C NMDARs reduces PPR variability in a manner dependent on astrocyte expression of <t>GluN1.</t> ( A–C ) Left: representative EPSC traces (average of 20 sweeps) to pairs of pulses (Δt = 50 ms) applied to Schaffer collateral axons in baseline and after wash-on of QNZ46 (25 µM). Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) before and after bath applying QNZ46 to control uninfected ( A ), Control ( B ) and Cre ( C ) slices. The p values were obtained for the population mean ( x - ) (paired sample t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients ( r ) and p values are shown in scatter plots. ( D–F ) Plots of EPSC amplitudes (normalized to the baseline average) before and during the application of QNZ46 to control uninfected ( D ), Control ( E ) and Cre-infected ( F ) slices (shaded area) where n is the number of inputs examined for each experiment. Baseline and experimental periods are indicated (black bars). Box plots show the summary of normalized EPSC amplitudes during the QNZ46 application. *p
    Guinea Pig Anti Nmdar1 Glun1 Extracellular Antibody, 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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    Alomone Labs anti trpm6 antibody
    <t>TRPM7/TRPM6</t> protein expression and TRPM7 kinase activity in splenic T cells. ( A ) Western blot analysis of immunoprecipitated TRPM7 from whole cell lysates of WT and KD splenic T cells. T cells were stimulated with PMA/ionomycin or anti-CD3/CD28 antibody coated beads for 48 hrs. Mouse embryonic fibroblasts were used as a positive control. Equal amounts of protein before immunoprecipitation were ensured by probing for actin. ( B ) Incorporation of 32 P into exogenous myelin basic protein (MBP) by TRPM7 immunoprecipitated from WT and KD resting T cells. Equal quantities of MBP were verified by coomassie blue staining. ( C ) Control experiment showing that anti-TRPM6 antibody was able to recognize TRPM6, by immunoprecipitation using anti-TRPM6 antibody in GFP-TRPM6 transfected HEK cells ( D .
    Anti Trpm6 Antibody, 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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    86
    millipore anti trpm6 antibody
    <t>TRPM7/TRPM6</t> protein expression and TRPM7 kinase activity in splenic T cells. ( A ) Western blot analysis of immunoprecipitated TRPM7 from whole cell lysates of WT and KD splenic T cells. T cells were stimulated with PMA/ionomycin or anti-CD3/CD28 antibody coated beads for 48 hrs. Mouse embryonic fibroblasts were used as a positive control. Equal amounts of protein before immunoprecipitation were ensured by probing for actin. ( B ) Incorporation of 32 P into exogenous myelin basic protein (MBP) by TRPM7 immunoprecipitated from WT and KD resting T cells. Equal quantities of MBP were verified by coomassie blue staining. ( C ) Control experiment showing that anti-TRPM6 antibody was able to recognize TRPM6, by immunoprecipitation using anti-TRPM6 antibody in GFP-TRPM6 transfected HEK cells ( D ). Western blot analysis of TRPM6 immunoprecipitated from WT and KD mouse T cells and kidneys. Full gel images are provided in Supplementary Fig. S6 .
    Anti Trpm6 Antibody, supplied by millipore, 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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    Inhibiting GluN2C NMDARs reduces PPR variability in a manner dependent on astrocyte expression of GluN1. ( A–C ) Left: representative EPSC traces (average of 20 sweeps) to pairs of pulses (Δt = 50 ms) applied to Schaffer collateral axons in baseline and after wash-on of QNZ46 (25 µM). Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) before and after bath applying QNZ46 to control uninfected ( A ), Control ( B ) and Cre ( C ) slices. The p values were obtained for the population mean ( x - ) (paired sample t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients ( r ) and p values are shown in scatter plots. ( D–F ) Plots of EPSC amplitudes (normalized to the baseline average) before and during the application of QNZ46 to control uninfected ( D ), Control ( E ) and Cre-infected ( F ) slices (shaded area) where n is the number of inputs examined for each experiment. Baseline and experimental periods are indicated (black bars). Box plots show the summary of normalized EPSC amplitudes during the QNZ46 application. *p

    Journal: eLife

    Article Title: Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum

    doi: 10.7554/eLife.70818

    Figure Lengend Snippet: Inhibiting GluN2C NMDARs reduces PPR variability in a manner dependent on astrocyte expression of GluN1. ( A–C ) Left: representative EPSC traces (average of 20 sweeps) to pairs of pulses (Δt = 50 ms) applied to Schaffer collateral axons in baseline and after wash-on of QNZ46 (25 µM). Right: PPR histograms (top) and ΔPPR vs. baseline PPR plots (bottom) before and after bath applying QNZ46 to control uninfected ( A ), Control ( B ) and Cre ( C ) slices. The p values were obtained for the population mean ( x - ) (paired sample t-test) and variance (δ 2 ) (one-tailed f-test for equal variances) comparing PPRs before and after the drug treatment. Linear fits and Pearson’s correlation coefficients ( r ) and p values are shown in scatter plots. ( D–F ) Plots of EPSC amplitudes (normalized to the baseline average) before and during the application of QNZ46 to control uninfected ( D ), Control ( E ) and Cre-infected ( F ) slices (shaded area) where n is the number of inputs examined for each experiment. Baseline and experimental periods are indicated (black bars). Box plots show the summary of normalized EPSC amplitudes during the QNZ46 application. *p

    Article Snippet: The primary antibodies used were chicken anti-RFP (1: 1000, Rockland #600-901-379), mouse anti-GFAP (1:1000, Synaptic Systems #173011), rabbit anti-GFAP (1:500, Abcam ab48050), rabbit anti-NeuN (1:500, Abcam #ab177487), and guinea pig anti-GluN1 (1:100, Alamone Labs AGP-046).

    Techniques: Expressing, One-tailed Test, Infection

    Knock-down of GluN1 in hippocampal astrocytes selectively alters the PPR disparity without affecting the basic properties of synaptic transmission. [Related also to Figure 6 ] ( A ) Left: Illustration of the experimental configuration of two-pathway stimulation approach to estimate PPR disparity in slices from Grin1 flx/flx mice infected with Control or Cre virus. Right: Comparison of ΔPPR (|PPR S1 -PPR S2 |) between Control or Cre slices. PPR disparity (ΔPPR) is reduced in Cre infected slices compared to controls. p = 0.009, Mann-Whitney U test. Control n = 29 pairs of inputs, 29 cells, 8 mice. Cre n = 28 pairs of inputs, 28 cells, 6 mice. ( B ) Left: Grand average waveforms of EPSCs recorded. Right: Box plots of EPSC risetime and EPSC decay time constant (single exponential fit). Each data point represents the average waveform of at least 5 sweeps of EPSCs. Control n = 46 inputs, 23 cells, 8 mice. Cre n = 41 inputs, 21 cells, 6 mice. ( C ) Left: Representative traces of spontaneous EPSC (sEPSC) recordings from Control (blue) or Cre (red) slices. Right: Boxplots of sEPSC amplitude and frequency measured in Control or Cre-infected slices. P values shown are the result of Mann-Whitney U test. Control n = 23 cells, 5 mice. Cre n = 26 cells, 5 mice. ( D–G ) Normalized sEPSC amplitude (top) and frequency (bottom) during baseline and in the presence of AP5 or QNZ46 in Control or Cre-infected slices. Neither sEPSC amplitude nor frequency is not affected by AP5 or QNZ46. Two tailed t-test or Mann-Whitney U test, comparisons made between Control vs. Cre normalized values in post drug period indicated. ( H–K ) ΔPPR (|PPR S1 -PPR S2 |) measurements during baseline and in the presence of AP5 or QNZ46 in Control or Cre-infected slices. The decrease in PPR disparity triggered by AP5 ( H ) and QNZ46 ( J ) is prevented upon knock-down of astrocyte NMDARs. * p

    Journal: eLife

    Article Title: Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum

    doi: 10.7554/eLife.70818

    Figure Lengend Snippet: Knock-down of GluN1 in hippocampal astrocytes selectively alters the PPR disparity without affecting the basic properties of synaptic transmission. [Related also to Figure 6 ] ( A ) Left: Illustration of the experimental configuration of two-pathway stimulation approach to estimate PPR disparity in slices from Grin1 flx/flx mice infected with Control or Cre virus. Right: Comparison of ΔPPR (|PPR S1 -PPR S2 |) between Control or Cre slices. PPR disparity (ΔPPR) is reduced in Cre infected slices compared to controls. p = 0.009, Mann-Whitney U test. Control n = 29 pairs of inputs, 29 cells, 8 mice. Cre n = 28 pairs of inputs, 28 cells, 6 mice. ( B ) Left: Grand average waveforms of EPSCs recorded. Right: Box plots of EPSC risetime and EPSC decay time constant (single exponential fit). Each data point represents the average waveform of at least 5 sweeps of EPSCs. Control n = 46 inputs, 23 cells, 8 mice. Cre n = 41 inputs, 21 cells, 6 mice. ( C ) Left: Representative traces of spontaneous EPSC (sEPSC) recordings from Control (blue) or Cre (red) slices. Right: Boxplots of sEPSC amplitude and frequency measured in Control or Cre-infected slices. P values shown are the result of Mann-Whitney U test. Control n = 23 cells, 5 mice. Cre n = 26 cells, 5 mice. ( D–G ) Normalized sEPSC amplitude (top) and frequency (bottom) during baseline and in the presence of AP5 or QNZ46 in Control or Cre-infected slices. Neither sEPSC amplitude nor frequency is not affected by AP5 or QNZ46. Two tailed t-test or Mann-Whitney U test, comparisons made between Control vs. Cre normalized values in post drug period indicated. ( H–K ) ΔPPR (|PPR S1 -PPR S2 |) measurements during baseline and in the presence of AP5 or QNZ46 in Control or Cre-infected slices. The decrease in PPR disparity triggered by AP5 ( H ) and QNZ46 ( J ) is prevented upon knock-down of astrocyte NMDARs. * p

    Article Snippet: The primary antibodies used were chicken anti-RFP (1: 1000, Rockland #600-901-379), mouse anti-GFAP (1:1000, Synaptic Systems #173011), rabbit anti-GFAP (1:500, Abcam ab48050), rabbit anti-NeuN (1:500, Abcam #ab177487), and guinea pig anti-GluN1 (1:100, Alamone Labs AGP-046).

    Techniques: Transmission Assay, Mouse Assay, Infection, MANN-WHITNEY, Two Tailed Test

    Adult mouse hippocampal astrocytes express GluN2C-NMDA receptors. ( A ) Top: patch RT-PCR strategy. Bottom: representative voltage responses to current steps in CA1 pyramidal neuron (far left, black), and current responses to voltage steps in astrocytes in stratum radiatum (SR, green), stratum oriens (SO, yellow), and stratum lacunosum moleculare (SLM, blue). Scale bars: neuron, 40 mV, 500 ms; astrocytes, 6 nA, 200 ms. ( B ) Summary of I-V curves of astrocytes obtained before collecting RNA. N = 39 cells, 36 slices, 9 mice for each layer. ( C ) Summary of RT-PCR analysis for mRNA encoding indicated NMDAR subunits: Grin1 (1), Grin2a (2A), Grin2b (2B), and Grin2c (2C). Control samples (no patch) were obtained by inserting the electrode into the slice without patching cells. Three technical replicates were performed for each condition. ( D ) Western blots of co-immunoprecipitations performed using adult mouse hippocampal extracts with the indicated antibodies where anti-Cre antibody is used as a negative control. The blots are probed for GluN2A/2B (top row), GluN2C (middle row) or GluN1 (bottom row). ( E ) Representative brain sections of Grin2c iCre/+ mice crossed to a tdTomato reporter line (Ai9), showing hippocampal area CA1 immunolabelled for GFAP, tdTomato and NeuN; scale bars, 160 μm (top), 25 μm (bottom). ( F ) Quantification of % cells that are positive for tdTomato amongst the counts of NeuN-labelled cells in stratum pyramidale (left, total n = 3155 cells) and SR (middle, total n = 619 cells), and GFAP-labelled cells (right, total n = 3149 cells). N = 10 fields of view of area CA1 from individual hippocampal sections (n = 10) obtained from 5 mice. Data are presented as mean ± s.e.m.

    Journal: eLife

    Article Title: Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum

    doi: 10.7554/eLife.70818

    Figure Lengend Snippet: Adult mouse hippocampal astrocytes express GluN2C-NMDA receptors. ( A ) Top: patch RT-PCR strategy. Bottom: representative voltage responses to current steps in CA1 pyramidal neuron (far left, black), and current responses to voltage steps in astrocytes in stratum radiatum (SR, green), stratum oriens (SO, yellow), and stratum lacunosum moleculare (SLM, blue). Scale bars: neuron, 40 mV, 500 ms; astrocytes, 6 nA, 200 ms. ( B ) Summary of I-V curves of astrocytes obtained before collecting RNA. N = 39 cells, 36 slices, 9 mice for each layer. ( C ) Summary of RT-PCR analysis for mRNA encoding indicated NMDAR subunits: Grin1 (1), Grin2a (2A), Grin2b (2B), and Grin2c (2C). Control samples (no patch) were obtained by inserting the electrode into the slice without patching cells. Three technical replicates were performed for each condition. ( D ) Western blots of co-immunoprecipitations performed using adult mouse hippocampal extracts with the indicated antibodies where anti-Cre antibody is used as a negative control. The blots are probed for GluN2A/2B (top row), GluN2C (middle row) or GluN1 (bottom row). ( E ) Representative brain sections of Grin2c iCre/+ mice crossed to a tdTomato reporter line (Ai9), showing hippocampal area CA1 immunolabelled for GFAP, tdTomato and NeuN; scale bars, 160 μm (top), 25 μm (bottom). ( F ) Quantification of % cells that are positive for tdTomato amongst the counts of NeuN-labelled cells in stratum pyramidale (left, total n = 3155 cells) and SR (middle, total n = 619 cells), and GFAP-labelled cells (right, total n = 3149 cells). N = 10 fields of view of area CA1 from individual hippocampal sections (n = 10) obtained from 5 mice. Data are presented as mean ± s.e.m.

    Article Snippet: The primary antibodies used were chicken anti-RFP (1: 1000, Rockland #600-901-379), mouse anti-GFAP (1:1000, Synaptic Systems #173011), rabbit anti-GFAP (1:500, Abcam ab48050), rabbit anti-NeuN (1:500, Abcam #ab177487), and guinea pig anti-GluN1 (1:100, Alamone Labs AGP-046).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Western Blot, Negative Control

    TRPM7/TRPM6 protein expression and TRPM7 kinase activity in splenic T cells. ( A ) Western blot analysis of immunoprecipitated TRPM7 from whole cell lysates of WT and KD splenic T cells. T cells were stimulated with PMA/ionomycin or anti-CD3/CD28 antibody coated beads for 48 hrs. Mouse embryonic fibroblasts were used as a positive control. Equal amounts of protein before immunoprecipitation were ensured by probing for actin. ( B ) Incorporation of 32 P into exogenous myelin basic protein (MBP) by TRPM7 immunoprecipitated from WT and KD resting T cells. Equal quantities of MBP were verified by coomassie blue staining. ( C ) Control experiment showing that anti-TRPM6 antibody was able to recognize TRPM6, by immunoprecipitation using anti-TRPM6 antibody in GFP-TRPM6 transfected HEK cells ( D .

    Journal: Scientific Reports

    Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry

    doi: 10.1038/s41598-018-21004-w

    Figure Lengend Snippet: TRPM7/TRPM6 protein expression and TRPM7 kinase activity in splenic T cells. ( A ) Western blot analysis of immunoprecipitated TRPM7 from whole cell lysates of WT and KD splenic T cells. T cells were stimulated with PMA/ionomycin or anti-CD3/CD28 antibody coated beads for 48 hrs. Mouse embryonic fibroblasts were used as a positive control. Equal amounts of protein before immunoprecipitation were ensured by probing for actin. ( B ) Incorporation of 32 P into exogenous myelin basic protein (MBP) by TRPM7 immunoprecipitated from WT and KD resting T cells. Equal quantities of MBP were verified by coomassie blue staining. ( C ) Control experiment showing that anti-TRPM6 antibody was able to recognize TRPM6, by immunoprecipitation using anti-TRPM6 antibody in GFP-TRPM6 transfected HEK cells ( D .

    Article Snippet: Extracts of T cells, mouse embryonic fibroblasts (MEF) or HEK293 cells (untransfected or heterologously expressing GFP tagged human TRPM6 in pEGFP-C1 vector) were obtained as described above for the TRPM7 kinase assay and incubated with anti-TRPM7 antibody (1:500) or anti-TRPM6 antibody (1:500, ACC-046; Alomone labs, Israel) overnight at 4 °C, followed by incubation with protein A sepharose beads for 1 hr.

    Techniques: Expressing, Activity Assay, Western Blot, Immunoprecipitation, Positive Control, Staining, Transfection

    Immunofluorescence staining of CBD-28k ( A ), TRPV5 ( B ), and TRPM6 ( C ) in renal tissue of wild-type and CBD-28k KO mice following CTZ and FSM treatment.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: The role of calbindin-D28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics

    doi: 10.1152/ajprenal.00057.2015

    Figure Lengend Snippet: Immunofluorescence staining of CBD-28k ( A ), TRPV5 ( B ), and TRPM6 ( C ) in renal tissue of wild-type and CBD-28k KO mice following CTZ and FSM treatment.

    Article Snippet: The membrane was incubated with an anti-TRPV5 antibody (1:1,000, Abcam), anti-CBD-28k antibody (1:20,000, Swant, Marly, Switzerland), anti-TRPM6 antibody (1:1,000, Alomone Laboratory, Jerusalem, Israel), and anti-CLDN16 antibody (1:3,000, Abcam) after blocking with 10% nonfat milk.

    Techniques: Immunofluorescence, Staining, Mouse Assay

    Immunoblotting study of CBD-28k ( A ) TRPV5 ( B ), TRPM6 ( C ), and claudin-16 ( D ) in renal tissue of wild-type and CBD-28k KO mice following CTZ and FSM treatment. The whole blots are shown with molecular size markers labeled on the left . The specific bands

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: The role of calbindin-D28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics

    doi: 10.1152/ajprenal.00057.2015

    Figure Lengend Snippet: Immunoblotting study of CBD-28k ( A ) TRPV5 ( B ), TRPM6 ( C ), and claudin-16 ( D ) in renal tissue of wild-type and CBD-28k KO mice following CTZ and FSM treatment. The whole blots are shown with molecular size markers labeled on the left . The specific bands

    Article Snippet: The membrane was incubated with an anti-TRPV5 antibody (1:1,000, Abcam), anti-CBD-28k antibody (1:20,000, Swant, Marly, Switzerland), anti-TRPM6 antibody (1:1,000, Alomone Laboratory, Jerusalem, Israel), and anti-CLDN16 antibody (1:3,000, Abcam) after blocking with 10% nonfat milk.

    Techniques: Mouse Assay, Labeling

    TRPM7/TRPM6 protein expression and TRPM7 kinase activity in splenic T cells. ( A ) Western blot analysis of immunoprecipitated TRPM7 from whole cell lysates of WT and KD splenic T cells. T cells were stimulated with PMA/ionomycin or anti-CD3/CD28 antibody coated beads for 48 hrs. Mouse embryonic fibroblasts were used as a positive control. Equal amounts of protein before immunoprecipitation were ensured by probing for actin. ( B ) Incorporation of 32 P into exogenous myelin basic protein (MBP) by TRPM7 immunoprecipitated from WT and KD resting T cells. Equal quantities of MBP were verified by coomassie blue staining. ( C ) Control experiment showing that anti-TRPM6 antibody was able to recognize TRPM6, by immunoprecipitation using anti-TRPM6 antibody in GFP-TRPM6 transfected HEK cells ( D ). Western blot analysis of TRPM6 immunoprecipitated from WT and KD mouse T cells and kidneys. Full gel images are provided in Supplementary Fig. S6 .

    Journal: Scientific Reports

    Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry

    doi: 10.1038/s41598-018-21004-w

    Figure Lengend Snippet: TRPM7/TRPM6 protein expression and TRPM7 kinase activity in splenic T cells. ( A ) Western blot analysis of immunoprecipitated TRPM7 from whole cell lysates of WT and KD splenic T cells. T cells were stimulated with PMA/ionomycin or anti-CD3/CD28 antibody coated beads for 48 hrs. Mouse embryonic fibroblasts were used as a positive control. Equal amounts of protein before immunoprecipitation were ensured by probing for actin. ( B ) Incorporation of 32 P into exogenous myelin basic protein (MBP) by TRPM7 immunoprecipitated from WT and KD resting T cells. Equal quantities of MBP were verified by coomassie blue staining. ( C ) Control experiment showing that anti-TRPM6 antibody was able to recognize TRPM6, by immunoprecipitation using anti-TRPM6 antibody in GFP-TRPM6 transfected HEK cells ( D ). Western blot analysis of TRPM6 immunoprecipitated from WT and KD mouse T cells and kidneys. Full gel images are provided in Supplementary Fig. S6 .

    Article Snippet: Extracts of T cells, mouse embryonic fibroblasts (MEF) or HEK293 cells (untransfected or heterologously expressing GFP tagged human TRPM6 in pEGFP-C1 vector) were obtained as described above for the TRPM7 kinase assay and incubated with anti-TRPM7 antibody (1:500) or anti-TRPM6 antibody (1:500, ACC-046; Alomone labs, Israel) overnight at 4 °C, followed by incubation with protein A sepharose beads for 1 hr.

    Techniques: Expressing, Activity Assay, Western Blot, Immunoprecipitation, Positive Control, Staining, Transfection