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

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

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

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

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

Journal: CNS Neuroscience & Therapeutics

Article Title: Disruption of the GABAergic system contributes to the development of perioperative neurocognitive disorders after anesthesia and surgery in aged mice, et al. Disruption of the GABAergic system contributes to the development of perioperative neurocognitive disorders after anesthesia and surgery in aged mice

doi: 10.1111/cns.13388

Figure Lengend Snippet: The expressions of neurotransmitters and different subunits of GABA A Rs. A‐B, The expression of GABA was decreased in the laparotomy mice, and no difference was found about glutamate (n = 9). C, The different average spectra of selected metabolites (GABA and glutamate). D‐F, The mRNA level of α5 subunit was up‐regulated at 1 day and continued to 10 days after laparotomy. No difference was found about the α1 and β3 subunits (n = 3). G‐I, The expressions of GAT‐3 and GAD65 were decreased, and the levels of surface α5GABA A Rs were increased in the laparotomy mice (n = 4). Data are presented as mean ± SEM. * P

Article Snippet: The antibodies used in this study include rabbit anti‐α5GABAA receptors, anti‐GAT‐3 (1:500‐1000; Alomone Labs), rabbit anti‐GAD65 (1:1000; Abcam), rabbit anti‐P38, p‐P38, ERK1/2, p‐ERK1/2, JNK1/2, p‐JNK1/2 (1:1000‐2000; Cell Signaling Technology), mouse anti‐GAPDH HRP‐conjugated goat‐anti‐mouse IgG, or anti‐rabbit IgG (1:1000‐5000; Promoter).

Techniques: Expressing, Mouse Assay