anti gaba transporter 1 gat 1 extracellular antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti gaba transporter 1 gat 1 extracellular antibody
    The <t>GABA</t> reuptake function of the mutant <t>GAT-1(Val125Met)</t> transporter was reduced in non-neuronal cells and neurons. A-B. HEK293T cells or mouse cortical astrocytes were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (1μg per a 35mm 2 dish) for 48 hrs before 3 H radioactive GABA uptake assay. C . Mouse cortical neurons were transfected with the wildtype or the mutant GAT-1(Val125Met) cDNAs at day 7 in culture. 3 H radioactive GABA uptake assay was performed after 8 days of transfection. GABA flux was measured after 30 min transport at room temperature. The influx of GABA, expressed in pmol/μg protein/min, was averaged from duplicates for each condition and for each transfection. The average counting was taken as n = 1. The untransfected condition was taken as baseline flux, which was subtracted from both the wildtype and the mutant conditions in HEK293T cells ( A ). The pmol/μg protein/min in the mutant was then normalized to the wildtype from each experiment, which was arbitrarily set as 100%. (***p
    Anti Gaba Transporter 1 Gat 1 Extracellular Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gaba transporter 1 gat 1 extracellular antibody/product/Alomone Labs
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    anti gaba transporter 1 gat 1 extracellular antibody - by Bioz Stars, 2022-12
    93/100 stars

    Images

    1) Product Images from "Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD"

    Article Title: Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD

    Journal: Experimental neurology

    doi: 10.1016/j.expneurol.2021.113723

    The GABA reuptake function of the mutant GAT-1(Val125Met) transporter was reduced in non-neuronal cells and neurons. A-B. HEK293T cells or mouse cortical astrocytes were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (1μg per a 35mm 2 dish) for 48 hrs before 3 H radioactive GABA uptake assay. C . Mouse cortical neurons were transfected with the wildtype or the mutant GAT-1(Val125Met) cDNAs at day 7 in culture. 3 H radioactive GABA uptake assay was performed after 8 days of transfection. GABA flux was measured after 30 min transport at room temperature. The influx of GABA, expressed in pmol/μg protein/min, was averaged from duplicates for each condition and for each transfection. The average counting was taken as n = 1. The untransfected condition was taken as baseline flux, which was subtracted from both the wildtype and the mutant conditions in HEK293T cells ( A ). The pmol/μg protein/min in the mutant was then normalized to the wildtype from each experiment, which was arbitrarily set as 100%. (***p
    Figure Legend Snippet: The GABA reuptake function of the mutant GAT-1(Val125Met) transporter was reduced in non-neuronal cells and neurons. A-B. HEK293T cells or mouse cortical astrocytes were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (1μg per a 35mm 2 dish) for 48 hrs before 3 H radioactive GABA uptake assay. C . Mouse cortical neurons were transfected with the wildtype or the mutant GAT-1(Val125Met) cDNAs at day 7 in culture. 3 H radioactive GABA uptake assay was performed after 8 days of transfection. GABA flux was measured after 30 min transport at room temperature. The influx of GABA, expressed in pmol/μg protein/min, was averaged from duplicates for each condition and for each transfection. The average counting was taken as n = 1. The untransfected condition was taken as baseline flux, which was subtracted from both the wildtype and the mutant conditions in HEK293T cells ( A ). The pmol/μg protein/min in the mutant was then normalized to the wildtype from each experiment, which was arbitrarily set as 100%. (***p

    Techniques Used: Mutagenesis, Transfection

    Electroencephalogram (EEG) of two sisters carrying the GAT-1(Val125Met) encoding mutation showing high amplitude burst of 3 Hz spike wave discharges and prominent occipital intermittent rhythmic delta activity (OIRDA). (A, B) Prominent bursts of Occipital Intermittent Rhythmic Delta Activity in the Proband (A, age 8) and Patient 2 (B, age 4) occurred throughout the waking state. (C, D) recordings showed sudden burst of generalized high amplitude bursts of 3.0 Hz spike and slow waves in proband ( C ) and the half-sister ( D ). ( E, F ), Typical absence for proband was associated with a myoclonic jerk and loss of tone—noted by muscle artifact (red arrows), sensitivity 15 microvolts (E) and a typical abnormal spike wave discharges for patient 2 ( D ) arising out of sleep, which was asymptomatic. Sensitivity 15 microvolts.
    Figure Legend Snippet: Electroencephalogram (EEG) of two sisters carrying the GAT-1(Val125Met) encoding mutation showing high amplitude burst of 3 Hz spike wave discharges and prominent occipital intermittent rhythmic delta activity (OIRDA). (A, B) Prominent bursts of Occipital Intermittent Rhythmic Delta Activity in the Proband (A, age 8) and Patient 2 (B, age 4) occurred throughout the waking state. (C, D) recordings showed sudden burst of generalized high amplitude bursts of 3.0 Hz spike and slow waves in proband ( C ) and the half-sister ( D ). ( E, F ), Typical absence for proband was associated with a myoclonic jerk and loss of tone—noted by muscle artifact (red arrows), sensitivity 15 microvolts (E) and a typical abnormal spike wave discharges for patient 2 ( D ) arising out of sleep, which was asymptomatic. Sensitivity 15 microvolts.

    Techniques Used: Mutagenesis, Activity Assay

    Both the total and cell surface expression of the mutant GAT-1(Val125Met) transporter protein was reduced. A, B . HEK293T cells were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (3μg/60 mm 2 ) for 48 hrs. The cells ( A ) were either harvested directly after wash with PBS for total lysates or followed by cell surface biotinylation to isolate the cell surface bound proteins ( B ). The total lysates ( A) or isolated surface protein ( B) were then analyzed by SDS-PAGE. Membranes were immunoblotted with rabbit anti-GAT-1 (1:300). ( C ). The total protein integrated density values (IDVs) from the total lysates ( C ) or isolated cell surface protein ( D ) were measured. The abundance of the mutant GAT-1(Val125Met) transporter was normalized to the wildtype cells expressing GAT-1 YFP . In C , the total protein abundance was measured by adding up all the bands between 90-110 KDa. The total protein IDVs of either the wildtype or the mutant was normalized to its loading control. The abundance of the mutant transporter was then normalized to the wildtype. (**p
    Figure Legend Snippet: Both the total and cell surface expression of the mutant GAT-1(Val125Met) transporter protein was reduced. A, B . HEK293T cells were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (3μg/60 mm 2 ) for 48 hrs. The cells ( A ) were either harvested directly after wash with PBS for total lysates or followed by cell surface biotinylation to isolate the cell surface bound proteins ( B ). The total lysates ( A) or isolated surface protein ( B) were then analyzed by SDS-PAGE. Membranes were immunoblotted with rabbit anti-GAT-1 (1:300). ( C ). The total protein integrated density values (IDVs) from the total lysates ( C ) or isolated cell surface protein ( D ) were measured. The abundance of the mutant GAT-1(Val125Met) transporter was normalized to the wildtype cells expressing GAT-1 YFP . In C , the total protein abundance was measured by adding up all the bands between 90-110 KDa. The total protein IDVs of either the wildtype or the mutant was normalized to its loading control. The abundance of the mutant transporter was then normalized to the wildtype. (**p

    Techniques Used: Expressing, Mutagenesis, Transfection, Isolation, SDS Page

    Modeling of the mutant GAT-1(Val125Met) protein with machine learning tools. ( A-B ). Tertiary structures of both the wildtype ( A ) and Val125Met mutant ( B ) GAT-1 protein are predicted by I-TASSER and DynaMut. The valine at residue 125 is mutated to methionine, both highlighted in light green, alongside with the surrounding residues. The interatomic interactions were predicted by DynaMut, where halogen bonds are depicted in blue and hydrogen bonds are colored in red. The Val125Met mutation results in the addition of sulfur into the sidechain, not causing drastic changes in polarity and charge, but making the protein less hydrophobic. ( C ). Machine learning tools predicted ΔΔG (Kcal/mol) of the mutant GAT-1(Val125Met) protein. Bars in the positive direction are predicted as stabilizing while bars in the negative direction are predicted as destabilizing.
    Figure Legend Snippet: Modeling of the mutant GAT-1(Val125Met) protein with machine learning tools. ( A-B ). Tertiary structures of both the wildtype ( A ) and Val125Met mutant ( B ) GAT-1 protein are predicted by I-TASSER and DynaMut. The valine at residue 125 is mutated to methionine, both highlighted in light green, alongside with the surrounding residues. The interatomic interactions were predicted by DynaMut, where halogen bonds are depicted in blue and hydrogen bonds are colored in red. The Val125Met mutation results in the addition of sulfur into the sidechain, not causing drastic changes in polarity and charge, but making the protein less hydrophobic. ( C ). Machine learning tools predicted ΔΔG (Kcal/mol) of the mutant GAT-1(Val125Met) protein. Bars in the positive direction are predicted as stabilizing while bars in the negative direction are predicted as destabilizing.

    Techniques Used: Mutagenesis

    2) Product Images from "GABA transporters regulate tonic and synaptic GABAA receptor-mediated currents in the suprachiasmatic nucleus neurons"

    Article Title: GABA transporters regulate tonic and synaptic GABAA receptor-mediated currents in the suprachiasmatic nucleus neurons

    Journal: Journal of Neurophysiology

    doi: 10.1152/jn.00194.2017

    Inhibition of GAT1 and GAT3 alters the circadian period of Per1 expression. A : representative bioluminescent records from the SCN of Per1:Luc cultured brain slices before and after application of SKF-89976A and SNAP-5114 (SKF+SNAP, 50 µM each; gray trace) or vehicle (DMSO, 0.1% final; black trace). The gray bar indicates the duration of application of the test agents. B : histogram of the circadian periods (means ± SE) measured before, during treatment with DMSO (0.1%), or during coapplication (SKF+SNAP, 50 µM each). * P
    Figure Legend Snippet: Inhibition of GAT1 and GAT3 alters the circadian period of Per1 expression. A : representative bioluminescent records from the SCN of Per1:Luc cultured brain slices before and after application of SKF-89976A and SNAP-5114 (SKF+SNAP, 50 µM each; gray trace) or vehicle (DMSO, 0.1% final; black trace). The gray bar indicates the duration of application of the test agents. B : histogram of the circadian periods (means ± SE) measured before, during treatment with DMSO (0.1%), or during coapplication (SKF+SNAP, 50 µM each). * P

    Techniques Used: Inhibition, Expressing, Cell Culture

    Effect of GAT1 and GAT3 inhibitors on sGPSC recorded in the SCN. A–L : changes in sGPSC parameters during single or joint SKF-89976A (SKF; 100 µM) and SNAP-5114 (SNAP; 100 µM) or nipecotic acid (NA; 2 mM) application. A, D, G, J : control, SKF ( n = 12), and SKF+SNAP ( n = 10). B, E, H, K : control, SNAP ( n = 10), and SNAP+SKF ( n = 9). C, F, I, L : control and NA ( n = 12). A–C : sGPSC recordings (each trace represents the average of 10 sGPSC recorded at each condition). D–F : amplitude. G–I : rise time (10–90%). J–L : decay time constant (tau). The sGPSC parameter changes between conditions are shown on the line graphs, which represent mean data for each recorded neuron (SE, which ranged from 4 to 6% of the mean, is not shown to make image clearer).
    Figure Legend Snippet: Effect of GAT1 and GAT3 inhibitors on sGPSC recorded in the SCN. A–L : changes in sGPSC parameters during single or joint SKF-89976A (SKF; 100 µM) and SNAP-5114 (SNAP; 100 µM) or nipecotic acid (NA; 2 mM) application. A, D, G, J : control, SKF ( n = 12), and SKF+SNAP ( n = 10). B, E, H, K : control, SNAP ( n = 10), and SNAP+SKF ( n = 9). C, F, I, L : control and NA ( n = 12). A–C : sGPSC recordings (each trace represents the average of 10 sGPSC recorded at each condition). D–F : amplitude. G–I : rise time (10–90%). J–L : decay time constant (tau). The sGPSC parameter changes between conditions are shown on the line graphs, which represent mean data for each recorded neuron (SE, which ranged from 4 to 6% of the mean, is not shown to make image clearer).

    Techniques Used:

    GAT1 inhibitors SKF-89976A and NNC-711 did not affect the expression of GAT1 and GAT3 in the hypothalamus. Brain slices were incubated for 5–7 h in ACSF containing SKF-89976A (SKF; 100 µM) dissolved in DMSO (0.1% final) or NNC-711 (NNC; 5 µM) dissolved in water. The brain tissue was then processed for Western blot analysis. A and B : effect of SKF; n = 6 triplicates: control, DMSO (vehicle), and SKF. C and D : effect of NNC-711; n = 8 duplicates: control and NNC-711. Bands above graphs show GAT1 ( A and C ) or GAT3 ( B and D ) expression and loading control (GAPDH). +Control, positive controls (cerebral cortex for GAT1 and thalamus for GAT3). The optical density of bands was quantified with ImageJ for each gel, normalized to the loading control, and shown as a ratio to control on bar graphs (means ± SE; one-way ANOVA, Tukey HSD post hoc test or t -test; NS, nonsignificant changes). GAT1 inhibitors did not change the expression of either GAT [ A : F 2,15 = 0.73 ( F crit = 3.68), P = 0.50; B : F 2,15 = 0.56 ( F crit = 3.68), P = 0.59; C : F 1,14 = 8E-05 ( F crit = 4.60), P = 0.99; D : F 1,14 = 1.43 ( F crit = 4.60), P = 0.25]. DMSO did not significantly affect the expression of the GAT proteins.
    Figure Legend Snippet: GAT1 inhibitors SKF-89976A and NNC-711 did not affect the expression of GAT1 and GAT3 in the hypothalamus. Brain slices were incubated for 5–7 h in ACSF containing SKF-89976A (SKF; 100 µM) dissolved in DMSO (0.1% final) or NNC-711 (NNC; 5 µM) dissolved in water. The brain tissue was then processed for Western blot analysis. A and B : effect of SKF; n = 6 triplicates: control, DMSO (vehicle), and SKF. C and D : effect of NNC-711; n = 8 duplicates: control and NNC-711. Bands above graphs show GAT1 ( A and C ) or GAT3 ( B and D ) expression and loading control (GAPDH). +Control, positive controls (cerebral cortex for GAT1 and thalamus for GAT3). The optical density of bands was quantified with ImageJ for each gel, normalized to the loading control, and shown as a ratio to control on bar graphs (means ± SE; one-way ANOVA, Tukey HSD post hoc test or t -test; NS, nonsignificant changes). GAT1 inhibitors did not change the expression of either GAT [ A : F 2,15 = 0.73 ( F crit = 3.68), P = 0.50; B : F 2,15 = 0.56 ( F crit = 3.68), P = 0.59; C : F 1,14 = 8E-05 ( F crit = 4.60), P = 0.99; D : F 1,14 = 1.43 ( F crit = 4.60), P = 0.25]. DMSO did not significantly affect the expression of the GAT proteins.

    Techniques Used: Expressing, Incubation, Western Blot

    Concentration dependence of GABA A R-mediated tonic current on the GAT1 and GAT3 inhibitors. A : concentration dependence of the inward tonic current activated by the GAT1 inhibitor SKF-89976A ( I ∆ = I (SKF+SNAP) − I SNAP , recording solution contained 20 µM SNAP-5114; whole cell recording from cerebral cortex; EC 50 = 32.6 ± 11.5 µM, n = 26 cells). B : concentration-response curve for GAT3 inhibitor SNAP-5114 ( I ∆ = I (SNAP+SKF) − I SKF , recording solution contained 10 µM SKF-89976A; recording from SCN; EC 50 = 79.0 ± 19.4 µM, n = 39 cells). On the y -axis, I ∆ (inward tonic current, in pA) is shown as positive numbers (means ± SE).
    Figure Legend Snippet: Concentration dependence of GABA A R-mediated tonic current on the GAT1 and GAT3 inhibitors. A : concentration dependence of the inward tonic current activated by the GAT1 inhibitor SKF-89976A ( I ∆ = I (SKF+SNAP) − I SNAP , recording solution contained 20 µM SNAP-5114; whole cell recording from cerebral cortex; EC 50 = 32.6 ± 11.5 µM, n = 26 cells). B : concentration-response curve for GAT3 inhibitor SNAP-5114 ( I ∆ = I (SNAP+SKF) − I SKF , recording solution contained 10 µM SKF-89976A; recording from SCN; EC 50 = 79.0 ± 19.4 µM, n = 39 cells). On the y -axis, I ∆ (inward tonic current, in pA) is shown as positive numbers (means ± SE).

    Techniques Used: Concentration Assay

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    Alomone Labs anti gaba transporter 1 gat 1 extracellular antibody
    The <t>GABA</t> reuptake function of the mutant <t>GAT-1(Val125Met)</t> transporter was reduced in non-neuronal cells and neurons. A-B. HEK293T cells or mouse cortical astrocytes were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (1μg per a 35mm 2 dish) for 48 hrs before 3 H radioactive GABA uptake assay. C . Mouse cortical neurons were transfected with the wildtype or the mutant GAT-1(Val125Met) cDNAs at day 7 in culture. 3 H radioactive GABA uptake assay was performed after 8 days of transfection. GABA flux was measured after 30 min transport at room temperature. The influx of GABA, expressed in pmol/μg protein/min, was averaged from duplicates for each condition and for each transfection. The average counting was taken as n = 1. The untransfected condition was taken as baseline flux, which was subtracted from both the wildtype and the mutant conditions in HEK293T cells ( A ). The pmol/μg protein/min in the mutant was then normalized to the wildtype from each experiment, which was arbitrarily set as 100%. (***p
    Anti Gaba Transporter 1 Gat 1 Extracellular Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gaba transporter 1 gat 1 extracellular antibody/product/Alomone Labs
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    anti gaba transporter 1 gat 1 extracellular antibody - by Bioz Stars, 2022-12
    93/100 stars
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    The GABA reuptake function of the mutant GAT-1(Val125Met) transporter was reduced in non-neuronal cells and neurons. A-B. HEK293T cells or mouse cortical astrocytes were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (1μg per a 35mm 2 dish) for 48 hrs before 3 H radioactive GABA uptake assay. C . Mouse cortical neurons were transfected with the wildtype or the mutant GAT-1(Val125Met) cDNAs at day 7 in culture. 3 H radioactive GABA uptake assay was performed after 8 days of transfection. GABA flux was measured after 30 min transport at room temperature. The influx of GABA, expressed in pmol/μg protein/min, was averaged from duplicates for each condition and for each transfection. The average counting was taken as n = 1. The untransfected condition was taken as baseline flux, which was subtracted from both the wildtype and the mutant conditions in HEK293T cells ( A ). The pmol/μg protein/min in the mutant was then normalized to the wildtype from each experiment, which was arbitrarily set as 100%. (***p

    Journal: Experimental neurology

    Article Title: Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD

    doi: 10.1016/j.expneurol.2021.113723

    Figure Lengend Snippet: The GABA reuptake function of the mutant GAT-1(Val125Met) transporter was reduced in non-neuronal cells and neurons. A-B. HEK293T cells or mouse cortical astrocytes were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (1μg per a 35mm 2 dish) for 48 hrs before 3 H radioactive GABA uptake assay. C . Mouse cortical neurons were transfected with the wildtype or the mutant GAT-1(Val125Met) cDNAs at day 7 in culture. 3 H radioactive GABA uptake assay was performed after 8 days of transfection. GABA flux was measured after 30 min transport at room temperature. The influx of GABA, expressed in pmol/μg protein/min, was averaged from duplicates for each condition and for each transfection. The average counting was taken as n = 1. The untransfected condition was taken as baseline flux, which was subtracted from both the wildtype and the mutant conditions in HEK293T cells ( A ). The pmol/μg protein/min in the mutant was then normalized to the wildtype from each experiment, which was arbitrarily set as 100%. (***p

    Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone Labs, AGT-001 or Synaptic System, 274102 at 1:200 dilution).

    Techniques: Mutagenesis, Transfection

    Electroencephalogram (EEG) of two sisters carrying the GAT-1(Val125Met) encoding mutation showing high amplitude burst of 3 Hz spike wave discharges and prominent occipital intermittent rhythmic delta activity (OIRDA). (A, B) Prominent bursts of Occipital Intermittent Rhythmic Delta Activity in the Proband (A, age 8) and Patient 2 (B, age 4) occurred throughout the waking state. (C, D) recordings showed sudden burst of generalized high amplitude bursts of 3.0 Hz spike and slow waves in proband ( C ) and the half-sister ( D ). ( E, F ), Typical absence for proband was associated with a myoclonic jerk and loss of tone—noted by muscle artifact (red arrows), sensitivity 15 microvolts (E) and a typical abnormal spike wave discharges for patient 2 ( D ) arising out of sleep, which was asymptomatic. Sensitivity 15 microvolts.

    Journal: Experimental neurology

    Article Title: Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD

    doi: 10.1016/j.expneurol.2021.113723

    Figure Lengend Snippet: Electroencephalogram (EEG) of two sisters carrying the GAT-1(Val125Met) encoding mutation showing high amplitude burst of 3 Hz spike wave discharges and prominent occipital intermittent rhythmic delta activity (OIRDA). (A, B) Prominent bursts of Occipital Intermittent Rhythmic Delta Activity in the Proband (A, age 8) and Patient 2 (B, age 4) occurred throughout the waking state. (C, D) recordings showed sudden burst of generalized high amplitude bursts of 3.0 Hz spike and slow waves in proband ( C ) and the half-sister ( D ). ( E, F ), Typical absence for proband was associated with a myoclonic jerk and loss of tone—noted by muscle artifact (red arrows), sensitivity 15 microvolts (E) and a typical abnormal spike wave discharges for patient 2 ( D ) arising out of sleep, which was asymptomatic. Sensitivity 15 microvolts.

    Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone Labs, AGT-001 or Synaptic System, 274102 at 1:200 dilution).

    Techniques: Mutagenesis, Activity Assay

    Both the total and cell surface expression of the mutant GAT-1(Val125Met) transporter protein was reduced. A, B . HEK293T cells were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (3μg/60 mm 2 ) for 48 hrs. The cells ( A ) were either harvested directly after wash with PBS for total lysates or followed by cell surface biotinylation to isolate the cell surface bound proteins ( B ). The total lysates ( A) or isolated surface protein ( B) were then analyzed by SDS-PAGE. Membranes were immunoblotted with rabbit anti-GAT-1 (1:300). ( C ). The total protein integrated density values (IDVs) from the total lysates ( C ) or isolated cell surface protein ( D ) were measured. The abundance of the mutant GAT-1(Val125Met) transporter was normalized to the wildtype cells expressing GAT-1 YFP . In C , the total protein abundance was measured by adding up all the bands between 90-110 KDa. The total protein IDVs of either the wildtype or the mutant was normalized to its loading control. The abundance of the mutant transporter was then normalized to the wildtype. (**p

    Journal: Experimental neurology

    Article Title: Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD

    doi: 10.1016/j.expneurol.2021.113723

    Figure Lengend Snippet: Both the total and cell surface expression of the mutant GAT-1(Val125Met) transporter protein was reduced. A, B . HEK293T cells were transfected with wildtype GAT-1 YFP (wt), or the mutant GAT-1(Val125Met, V125M) YFP cDNAs (3μg/60 mm 2 ) for 48 hrs. The cells ( A ) were either harvested directly after wash with PBS for total lysates or followed by cell surface biotinylation to isolate the cell surface bound proteins ( B ). The total lysates ( A) or isolated surface protein ( B) were then analyzed by SDS-PAGE. Membranes were immunoblotted with rabbit anti-GAT-1 (1:300). ( C ). The total protein integrated density values (IDVs) from the total lysates ( C ) or isolated cell surface protein ( D ) were measured. The abundance of the mutant GAT-1(Val125Met) transporter was normalized to the wildtype cells expressing GAT-1 YFP . In C , the total protein abundance was measured by adding up all the bands between 90-110 KDa. The total protein IDVs of either the wildtype or the mutant was normalized to its loading control. The abundance of the mutant transporter was then normalized to the wildtype. (**p

    Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone Labs, AGT-001 or Synaptic System, 274102 at 1:200 dilution).

    Techniques: Expressing, Mutagenesis, Transfection, Isolation, SDS Page

    Modeling of the mutant GAT-1(Val125Met) protein with machine learning tools. ( A-B ). Tertiary structures of both the wildtype ( A ) and Val125Met mutant ( B ) GAT-1 protein are predicted by I-TASSER and DynaMut. The valine at residue 125 is mutated to methionine, both highlighted in light green, alongside with the surrounding residues. The interatomic interactions were predicted by DynaMut, where halogen bonds are depicted in blue and hydrogen bonds are colored in red. The Val125Met mutation results in the addition of sulfur into the sidechain, not causing drastic changes in polarity and charge, but making the protein less hydrophobic. ( C ). Machine learning tools predicted ΔΔG (Kcal/mol) of the mutant GAT-1(Val125Met) protein. Bars in the positive direction are predicted as stabilizing while bars in the negative direction are predicted as destabilizing.

    Journal: Experimental neurology

    Article Title: Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD

    doi: 10.1016/j.expneurol.2021.113723

    Figure Lengend Snippet: Modeling of the mutant GAT-1(Val125Met) protein with machine learning tools. ( A-B ). Tertiary structures of both the wildtype ( A ) and Val125Met mutant ( B ) GAT-1 protein are predicted by I-TASSER and DynaMut. The valine at residue 125 is mutated to methionine, both highlighted in light green, alongside with the surrounding residues. The interatomic interactions were predicted by DynaMut, where halogen bonds are depicted in blue and hydrogen bonds are colored in red. The Val125Met mutation results in the addition of sulfur into the sidechain, not causing drastic changes in polarity and charge, but making the protein less hydrophobic. ( C ). Machine learning tools predicted ΔΔG (Kcal/mol) of the mutant GAT-1(Val125Met) protein. Bars in the positive direction are predicted as stabilizing while bars in the negative direction are predicted as destabilizing.

    Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone Labs, AGT-001 or Synaptic System, 274102 at 1:200 dilution).

    Techniques: Mutagenesis

    Inhibition of GAT1 and GAT3 alters the circadian period of Per1 expression. A : representative bioluminescent records from the SCN of Per1:Luc cultured brain slices before and after application of SKF-89976A and SNAP-5114 (SKF+SNAP, 50 µM each; gray trace) or vehicle (DMSO, 0.1% final; black trace). The gray bar indicates the duration of application of the test agents. B : histogram of the circadian periods (means ± SE) measured before, during treatment with DMSO (0.1%), or during coapplication (SKF+SNAP, 50 µM each). * P

    Journal: Journal of Neurophysiology

    Article Title: GABA transporters regulate tonic and synaptic GABAA receptor-mediated currents in the suprachiasmatic nucleus neurons

    doi: 10.1152/jn.00194.2017

    Figure Lengend Snippet: Inhibition of GAT1 and GAT3 alters the circadian period of Per1 expression. A : representative bioluminescent records from the SCN of Per1:Luc cultured brain slices before and after application of SKF-89976A and SNAP-5114 (SKF+SNAP, 50 µM each; gray trace) or vehicle (DMSO, 0.1% final; black trace). The gray bar indicates the duration of application of the test agents. B : histogram of the circadian periods (means ± SE) measured before, during treatment with DMSO (0.1%), or during coapplication (SKF+SNAP, 50 µM each). * P

    Article Snippet: For the enhanced chemiluminescence detection, the blots were blocked in 5% non-fat milk in TBST (Tris-buffered saline-Tween 20) and incubated with a rabbit anti-GAT1 (extracellular domain) polyclonal antibody (AGT-001; Alomone Laboratories, Jerusalem, Israel 1:500) overnight at 4°C in the same buffer with agitation.

    Techniques: Inhibition, Expressing, Cell Culture

    Effect of GAT1 and GAT3 inhibitors on sGPSC recorded in the SCN. A–L : changes in sGPSC parameters during single or joint SKF-89976A (SKF; 100 µM) and SNAP-5114 (SNAP; 100 µM) or nipecotic acid (NA; 2 mM) application. A, D, G, J : control, SKF ( n = 12), and SKF+SNAP ( n = 10). B, E, H, K : control, SNAP ( n = 10), and SNAP+SKF ( n = 9). C, F, I, L : control and NA ( n = 12). A–C : sGPSC recordings (each trace represents the average of 10 sGPSC recorded at each condition). D–F : amplitude. G–I : rise time (10–90%). J–L : decay time constant (tau). The sGPSC parameter changes between conditions are shown on the line graphs, which represent mean data for each recorded neuron (SE, which ranged from 4 to 6% of the mean, is not shown to make image clearer).

    Journal: Journal of Neurophysiology

    Article Title: GABA transporters regulate tonic and synaptic GABAA receptor-mediated currents in the suprachiasmatic nucleus neurons

    doi: 10.1152/jn.00194.2017

    Figure Lengend Snippet: Effect of GAT1 and GAT3 inhibitors on sGPSC recorded in the SCN. A–L : changes in sGPSC parameters during single or joint SKF-89976A (SKF; 100 µM) and SNAP-5114 (SNAP; 100 µM) or nipecotic acid (NA; 2 mM) application. A, D, G, J : control, SKF ( n = 12), and SKF+SNAP ( n = 10). B, E, H, K : control, SNAP ( n = 10), and SNAP+SKF ( n = 9). C, F, I, L : control and NA ( n = 12). A–C : sGPSC recordings (each trace represents the average of 10 sGPSC recorded at each condition). D–F : amplitude. G–I : rise time (10–90%). J–L : decay time constant (tau). The sGPSC parameter changes between conditions are shown on the line graphs, which represent mean data for each recorded neuron (SE, which ranged from 4 to 6% of the mean, is not shown to make image clearer).

    Article Snippet: For the enhanced chemiluminescence detection, the blots were blocked in 5% non-fat milk in TBST (Tris-buffered saline-Tween 20) and incubated with a rabbit anti-GAT1 (extracellular domain) polyclonal antibody (AGT-001; Alomone Laboratories, Jerusalem, Israel 1:500) overnight at 4°C in the same buffer with agitation.

    Techniques:

    GAT1 inhibitors SKF-89976A and NNC-711 did not affect the expression of GAT1 and GAT3 in the hypothalamus. Brain slices were incubated for 5–7 h in ACSF containing SKF-89976A (SKF; 100 µM) dissolved in DMSO (0.1% final) or NNC-711 (NNC; 5 µM) dissolved in water. The brain tissue was then processed for Western blot analysis. A and B : effect of SKF; n = 6 triplicates: control, DMSO (vehicle), and SKF. C and D : effect of NNC-711; n = 8 duplicates: control and NNC-711. Bands above graphs show GAT1 ( A and C ) or GAT3 ( B and D ) expression and loading control (GAPDH). +Control, positive controls (cerebral cortex for GAT1 and thalamus for GAT3). The optical density of bands was quantified with ImageJ for each gel, normalized to the loading control, and shown as a ratio to control on bar graphs (means ± SE; one-way ANOVA, Tukey HSD post hoc test or t -test; NS, nonsignificant changes). GAT1 inhibitors did not change the expression of either GAT [ A : F 2,15 = 0.73 ( F crit = 3.68), P = 0.50; B : F 2,15 = 0.56 ( F crit = 3.68), P = 0.59; C : F 1,14 = 8E-05 ( F crit = 4.60), P = 0.99; D : F 1,14 = 1.43 ( F crit = 4.60), P = 0.25]. DMSO did not significantly affect the expression of the GAT proteins.

    Journal: Journal of Neurophysiology

    Article Title: GABA transporters regulate tonic and synaptic GABAA receptor-mediated currents in the suprachiasmatic nucleus neurons

    doi: 10.1152/jn.00194.2017

    Figure Lengend Snippet: GAT1 inhibitors SKF-89976A and NNC-711 did not affect the expression of GAT1 and GAT3 in the hypothalamus. Brain slices were incubated for 5–7 h in ACSF containing SKF-89976A (SKF; 100 µM) dissolved in DMSO (0.1% final) or NNC-711 (NNC; 5 µM) dissolved in water. The brain tissue was then processed for Western blot analysis. A and B : effect of SKF; n = 6 triplicates: control, DMSO (vehicle), and SKF. C and D : effect of NNC-711; n = 8 duplicates: control and NNC-711. Bands above graphs show GAT1 ( A and C ) or GAT3 ( B and D ) expression and loading control (GAPDH). +Control, positive controls (cerebral cortex for GAT1 and thalamus for GAT3). The optical density of bands was quantified with ImageJ for each gel, normalized to the loading control, and shown as a ratio to control on bar graphs (means ± SE; one-way ANOVA, Tukey HSD post hoc test or t -test; NS, nonsignificant changes). GAT1 inhibitors did not change the expression of either GAT [ A : F 2,15 = 0.73 ( F crit = 3.68), P = 0.50; B : F 2,15 = 0.56 ( F crit = 3.68), P = 0.59; C : F 1,14 = 8E-05 ( F crit = 4.60), P = 0.99; D : F 1,14 = 1.43 ( F crit = 4.60), P = 0.25]. DMSO did not significantly affect the expression of the GAT proteins.

    Article Snippet: For the enhanced chemiluminescence detection, the blots were blocked in 5% non-fat milk in TBST (Tris-buffered saline-Tween 20) and incubated with a rabbit anti-GAT1 (extracellular domain) polyclonal antibody (AGT-001; Alomone Laboratories, Jerusalem, Israel 1:500) overnight at 4°C in the same buffer with agitation.

    Techniques: Expressing, Incubation, Western Blot

    Concentration dependence of GABA A R-mediated tonic current on the GAT1 and GAT3 inhibitors. A : concentration dependence of the inward tonic current activated by the GAT1 inhibitor SKF-89976A ( I ∆ = I (SKF+SNAP) − I SNAP , recording solution contained 20 µM SNAP-5114; whole cell recording from cerebral cortex; EC 50 = 32.6 ± 11.5 µM, n = 26 cells). B : concentration-response curve for GAT3 inhibitor SNAP-5114 ( I ∆ = I (SNAP+SKF) − I SKF , recording solution contained 10 µM SKF-89976A; recording from SCN; EC 50 = 79.0 ± 19.4 µM, n = 39 cells). On the y -axis, I ∆ (inward tonic current, in pA) is shown as positive numbers (means ± SE).

    Journal: Journal of Neurophysiology

    Article Title: GABA transporters regulate tonic and synaptic GABAA receptor-mediated currents in the suprachiasmatic nucleus neurons

    doi: 10.1152/jn.00194.2017

    Figure Lengend Snippet: Concentration dependence of GABA A R-mediated tonic current on the GAT1 and GAT3 inhibitors. A : concentration dependence of the inward tonic current activated by the GAT1 inhibitor SKF-89976A ( I ∆ = I (SKF+SNAP) − I SNAP , recording solution contained 20 µM SNAP-5114; whole cell recording from cerebral cortex; EC 50 = 32.6 ± 11.5 µM, n = 26 cells). B : concentration-response curve for GAT3 inhibitor SNAP-5114 ( I ∆ = I (SNAP+SKF) − I SKF , recording solution contained 10 µM SKF-89976A; recording from SCN; EC 50 = 79.0 ± 19.4 µM, n = 39 cells). On the y -axis, I ∆ (inward tonic current, in pA) is shown as positive numbers (means ± SE).

    Article Snippet: For the enhanced chemiluminescence detection, the blots were blocked in 5% non-fat milk in TBST (Tris-buffered saline-Tween 20) and incubated with a rabbit anti-GAT1 (extracellular domain) polyclonal antibody (AGT-001; Alomone Laboratories, Jerusalem, Israel 1:500) overnight at 4°C in the same buffer with agitation.

    Techniques: Concentration Assay