gaba r β3  (NeuroMab)

Journal: EMBO Reports

Article Title: miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity

doi: 10.1038/s44319-024-00253-z

Figure Lengend Snippet: ( A ) Schematic of RNA-Induced Silencing Complex (RISC) and miR153 interaction with the seed site in Gphn 3’UTR, which is predicted to suppress translation of this mRNA. ( B ) qRT-PCR of miR153 and miR15a (Ctrl miRNA) expression in cultured hippocampal neurons harvested at different time-points following iLTP stimulation. miRNA levels were normalized to U6. N = 4. P -values (Ctrl vs iLTP t = 10/20/45/90 min): miR15a = 0.3113/0.8460/0.6049/0.3843, miR153 = 0.3811/0.8735/0.0040/<0.0001 ( C ) Schematic of the Luc- Gphn luciferase reporters. miR153 seed site is mutated in Luc- Gphn 153-Mut . ( D ) Quantification of Luc- Gphn activities in HEK293T cells co-expressing control miRNA (miRCon), miR153, or no miRNA. Firefly was normalized to Renilla, and the data quantified as relative change in normalized Luc activity. N = 5. P -values: WT no miRNA vs miR153 = 0.0005, WT miRCon vs miR153 = 0.0002, Mut no miRNA vs miR153 = 0.8692, Mut miRCon vs miR153 = 0.9018, WT miR153 vs Mut miR153 < 0.0001. ( E ) Quantification of Luc- Gphn activities in hippocampal neurons under control conditions (Ctrl) or 90 min post-iLTP stimulation. N = 6. P -values: WT Ctrl vs iLTP = 0.0009, WT Ctrl vs Mut Ctrl = 0.0249, WT Ctrl vs Mut iLTP = 0.0007, Mut Ctrl vs Mut iLTP = 0.4386. ( F ) Western blots of gephyrin (GPHN), GABA A R subunits α1 and γ2, GAPDH, and GFP protein levels in neurons overexpressing miRCon or miR153 (left), and miRCon inhibitor or miR153 inhibitor (right). miRNA overexpression (OE) constructs contain a GFP reporter. ( G ) Quantification of GPHN, α1, and γ2 levels in miRCon or miR153 OE neurons. Protein levels were normalized to GAPDH, and the data quantified as relative change in normalized protein expression. N = 5. P -values (miRCon vs miR153): GPHN = 0.0079, α1 = 0.1508, γ2 = 0.3095. ( H ) Quantification of GPHN, α1, and γ2 in neurons expressing miRCon or miR153 inhibitors. N = 4. P -values (anti-Con vs anti-153): GPHN = 0.0286, α1 = 0.2000, γ2 = 0.2000. N = independent neuronal cultures/experiments. All values represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001; one-sample t-test ( B ), two-way ANOVA with Tukey’s ( D ) or Šidák’s ( E ) multiple comparisons post-hoc test, and Mann–Whitney test ( G , H ).

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate primary antibody: GPHN (1:5000 Synaptic Systems 147111), GABA A Rα1 (1:1000 NeuroMab 75136), GABA A Rγ2 (1:1000 NeuroMab 75442), GAPDH (1:10,000 GeneTex 627408), GFP (1:2000 NeuroMab 75131), GABA A R β3 (1:1000 NeuroMab 75149), GABA A Rα5 (1:1000 NeuroMab 455510), GluA1 (1:1000 Millipore ABN241), NL2 (1:2500 Synaptic Systems 129511), CB (1:1000 Synaptic Systems 261 003), VAMP2 (1:5000 Synaptic Systems 104211).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Luciferase, Control, Activity Assay, Western Blot, Over Expression, Construct, MANN-WHITNEY

Journal: EMBO Reports

Article Title: miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity

doi: 10.1038/s44319-024-00253-z

Figure Lengend Snippet: ( A ) Western blot (WB) of AGO2 immunoprecipitated from neurons following control treatment (Ctrl) or 90 min post-iLTP stimulation (iLTP). ( B ) qRT-PCR of Gphn mRNA bound to AGO2 in neurons from ( A ). AGO2-bound Gphn was normalized to total Gphn mRNA expression, and fold change from Ctrl was quantified for each condition. N = 4. P = 0.0452. ( C ) qRT-PCR of total Gphn mRNA levels in Ctrl and iLTP-90 neurons. Gphn mRNA levels were normalized to U6 expression, and fold change from Ctrl was quantified for each condition. N = 3. P = 0.7500. ( D ) Left: western blots of GABA A R subunits α5 (extrasynaptic) and β3 (synaptic), AMPAR subunit GluA1, GPHN binding proteins neuroligin-2 (NL2) and collybistin (CB), miR153 target VAMP2, GAPDH, and GFP protein levels in neurons overexpressing miRCon or miR153miRNA overexpression (OE) constructs contain a GFP reporter. Right: quantification of α5, β3, GluA1, NL2, CB, VAMP2 in miRCon or miR153 OE neurons. Protein levels were normalized to GAPDH, and the data quantified as relative change in normalized protein expression. N = 5. P -values (miRCon vs miR153): α5 > 0.9999, β3 = 0.5476, GluA1 = 0.5476, NL2 = 0.4206, CB > 0.9999, VAMP2 = 0.0286. ( E ) Left: western blots of α5, β3, GluA1, NL2, VAMP2, and GAPDH protein levels in neurons expressing miRCon or miR153 inhibitors. Right: quantification of α5, β3, GluA1, NL2, CB, VAMP2 in miRCon neurons or neurons in which miR153 was inhibited. Protein levels were normalized to GAPDH, and the data quantified as relative change in normalized protein expression. N = 4. P -values (anti-Con vs anti-153): α5 = 0.8857, β3 = 0.8857, GluA1 = 0.8857, NL2 = 0.8857, CB > 0.9999, VAMP2 = 0.6857. N = independent neuronal cultures/experiments. All values represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001; one-sample t-test ( B ), Wilcoxon signed rank test ( C ), and Mann–Whitney test ( D , E ).

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate primary antibody: GPHN (1:5000 Synaptic Systems 147111), GABA A Rα1 (1:1000 NeuroMab 75136), GABA A Rγ2 (1:1000 NeuroMab 75442), GAPDH (1:10,000 GeneTex 627408), GFP (1:2000 NeuroMab 75131), GABA A R β3 (1:1000 NeuroMab 75149), GABA A Rα5 (1:1000 NeuroMab 455510), GluA1 (1:1000 Millipore ABN241), NL2 (1:2500 Synaptic Systems 129511), CB (1:1000 Synaptic Systems 261 003), VAMP2 (1:5000 Synaptic Systems 104211).

Techniques: Western Blot, Immunoprecipitation, Control, Quantitative RT-PCR, Expressing, Binding Assay, Over Expression, Construct, MANN-WHITNEY

Journal: EMBO Reports

Article Title: miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity

doi: 10.1038/s44319-024-00253-z

Figure Lengend Snippet: ( A ) Representative dendritic segments of miRCon or miR153 OE-expressing neurons over time in control and iLTP conditions. Neurons co-expressed the gephyrin intrabody (GPHN IB, arrowheads) and labeled live with VGAT Oyster 650 . Puncta are labeled with filled arrowheads when the fluorescence is unchanged and open arrowheads when fluorescence increases over time. Boxes indicate the fluorescent puncta enlarged in the merged images (dendrite scale bar, 10 μm; synapse scale bar, 2 μm). ( B ) Quantification of fold change in GPHN puncta fluorescence intensity over time following treatment in neurons from ( A ). N = 3 / n = 15 neurons per condition. P -values (miRCon Ctrl vs iLTP): 10 min > 0.9999, 20 min = 0.0125, 30 min = 0.0049, 45 min = 0.0012, 60 min < 0.0001, 90 min < 0.0001. ( C ) Paired measurements of GPHN cluster density in dendrites prior to (−5 min) and 90 min following treatment. N = 3 / n = 15 neurons per condition. P -values ( t = −5 min vs t = 90 min): GPHN density miRCon Ctrl = 0.9137, GPHN density miRCon iLTP < 0.0001, GPHN density miR153 Ctrl = 0.9965, GPHN density miR153 iLTP = 0.9999. ( D ) Representative dendritic segments of miRCon or miR153 OE-expressing neurons labeled with antibodies to surface GABA A R γ2 subunit (sGABA A R) and VGAT following control treatment or 90 min post-iLTP stimulation. Scale bar, 10 μm. ( E ) Quantification of sGABA A R and VGAT cluster area (left) and density (right) in neurons from ( D ). N = 3/ n = 27–36 neurons per condition. P-values: γ2 area miRCon Ctrl vs iLTP = 0.0270, γ2 area miRCon Ctrl vs miR153 Ctrl = 0.0110, γ2 area miRCon Ctrl vs miR153 iLTP = 0.0040, γ2 area miRCon iLTP vs miR153 Ctrl < 0.0001, γ2 area miRCon iLTP vs miR153 iLTP < 0.0001, γ2 area miR153 Ctrl vs iLTP = 0.9985; VGAT area miRCon Ctrl vs iLTP = 0.0179, VGAT area miRCon Ctrl vs miR153 Ctrl = 0.0016, VGAT area miRCon Ctrl vs miR153 iLTP = 0.0024, VGAT area miRCon iLTP vs miR153 Ctrl < 0.0001, VGAT area miRCon iLTP vs miR153 iLTP < 0.0001, VGAT area miR153 Ctrl vs iLTP > 0.9999; γ2 density miRCon Ctrl vs iLTP < 0.0001, γ2 density miRCon Ctrl vs miR153 Ctrl = 0.0016, γ2 density miRCon Ctrl vs miR153 iLTP = 0.0006, γ2 density miRCon iLTP vs miR153 Ctrl < 0.0001, γ2 density miRCon iLTP vs miR153 iLTP < 0.0001, γ2 density miR153 Ctrl vs iLTP = 0.9989; VGAT density miRCon Ctrl vs iLTP < 0.0001, VGAT density miRCon Ctrl vs miR153 Ctrl < 0.0001, VGAT density miRCon Ctrl vs miR153 iLTP < 0.0001, VGAT density miRCon iLTP vs miR153 Ctrl < 0.0001, VGAT density miRCon iLTP vs miR153 iLTP < 0.0001, VGAT density miR153 Ctrl vs iLTP = 0.9986. N = independent neuronal cultures/experiments, n = neurons. All values represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001; mixed-effects model with Geisser-Greenhouse correction ( B ) and Šidák’s multiple comparisons post-hoc test ( B , C ) and ordinary two-way ANOVA with Tukey’s multiple comparisons post-hoc test ( E ).

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate primary antibody: GPHN (1:5000 Synaptic Systems 147111), GABA A Rα1 (1:1000 NeuroMab 75136), GABA A Rγ2 (1:1000 NeuroMab 75442), GAPDH (1:10,000 GeneTex 627408), GFP (1:2000 NeuroMab 75131), GABA A R β3 (1:1000 NeuroMab 75149), GABA A Rα5 (1:1000 NeuroMab 455510), GluA1 (1:1000 Millipore ABN241), NL2 (1:2500 Synaptic Systems 129511), CB (1:1000 Synaptic Systems 261 003), VAMP2 (1:5000 Synaptic Systems 104211).

Techniques: Expressing, Control, Labeling, Fluorescence

Journal: EMBO Reports

Article Title: miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity

doi: 10.1038/s44319-024-00253-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate primary antibody: GPHN (1:5000 Synaptic Systems 147111), GABA A Rα1 (1:1000 NeuroMab 75136), GABA A Rγ2 (1:1000 NeuroMab 75442), GAPDH (1:10,000 GeneTex 627408), GFP (1:2000 NeuroMab 75131), GABA A R β3 (1:1000 NeuroMab 75149), GABA A Rα5 (1:1000 NeuroMab 455510), GluA1 (1:1000 Millipore ABN241), NL2 (1:2500 Synaptic Systems 129511), CB (1:1000 Synaptic Systems 261 003), VAMP2 (1:5000 Synaptic Systems 104211).

Techniques: Recombinant, Control, Sequencing, Clone Assay, Software, Reporter Assay, Western Blot

Journal: Biomolecules

Article Title: Epileptic Encephalopathy GABRB Structural Variants Share Common Gating and Trafficking Defects.

doi: 10.3390/biom13121790

Figure Lengend Snippet: Figure 3. GABRB3 mutations altered the stability of GABAA receptors. (A) Cryo-EM structure of the pentameric α1β3γ2 GABAA receptor (6HUP) viewed from the side, with the β subunits in red, the α subunits in blue, and the γ subunit in gray. GABRB3 mutations are mapped onto the structure and represented as green sticks and numbered according to the panels in (B). Network of interatomic interactions predicted by wild-type (wt, left panels) and β3 mutant (mut, right panels) 6HUP receptors. The panels are numbered according to the labels in panel (A), where the mutations are mapped. wt and mut amino acids are colored in light green, and the neighborhood residues are in CPK representation. All residues are represented as sticks. In brackets, the residues are numbered based on the 6HUP structure. For correspondence with the GABRB3 protein sequence deposited in UniProt-P28472, 25 must be added to the number indicated on the panel. Structural domains involved in the network of interactions are labeled, and residues are equally colored. ∆∆G values are shown on the corresponding panels.

Article Snippet: The primary antibodies used to detect GABAA receptors were as follows: mouse anti-α1 subunit antibody (1:500; NeuroMab, 75-136, Davis, CA, USA), rabbit anti-β3 subunit antibody (1:500; Novus, NB300199, St. Louis, MO, USA), rabbit anti-β2 subunit antibody (1:1000; Millipore, AB5561, Burlington, MA, USA), and rabbit anti-γ2 subunit antibody (1:1000; Millipore, AB5559).

Techniques: Cryo-EM Sample Prep, Mutagenesis, Sequencing, Labeling

Journal: Biomolecules

Article Title: Epileptic Encephalopathy GABRB Structural Variants Share Common Gating and Trafficking Defects.

doi: 10.3390/biom13121790

Figure Lengend Snippet: Figure 5. GABRB3 mutations altered the flexibility and function of GABAA receptors. (A) Structural representation of the GABAA receptor flexible conformation based on the vibrational entropy energy change (∆∆SVib ENCoM) predicted between the wild-type and the mutant β3 subunit in the 6HUP structure. Mutant β3 subunits are colored according to the vibrational entropy perturbation upon mutation. Shades of blue represent a loss of flexibility (rigidification of the structure), and shades of red mean an increase in structural flexibility. ∆∆SVib ENCoM values are shown at the bottom of the corresponding mutation. In brackets, the variants are numbered based on the 6HUP structure. (B) In the upper panel, concentration–response curves of wild-type and mutant α1β3γ2 receptors are shown. GABA-evoked currents were normalized to the maximal response to 1 mM GABA. Lower panel, Pearson correlation coefficients obtained from the ∆∆G of the mutant β3 structures were plotted against surface (left graph) and total (right graph) expression levels of α1, β3, and γ2 subunits according to the data displayed in panels (C,D), respectively. Lines represent linear regression fits. (C,D) Surface and total expression levels of wild-type and mutant β3 subunits coexpressed with α1 and γ2 subunits, respectively.

Article Snippet: The primary antibodies used to detect GABAA receptors were as follows: mouse anti-α1 subunit antibody (1:500; NeuroMab, 75-136, Davis, CA, USA), rabbit anti-β3 subunit antibody (1:500; Novus, NB300199, St. Louis, MO, USA), rabbit anti-β2 subunit antibody (1:1000; Millipore, AB5561, Burlington, MA, USA), and rabbit anti-γ2 subunit antibody (1:1000; Millipore, AB5559).

Techniques: Mutagenesis, Concentration Assay, Expressing

Journal: iScience

Article Title: Distinct mechanisms drive sequential internalization and degradation of GABA A Rs during global ischemia and reperfusion injury

doi: 10.1016/j.isci.2023.108061

Figure Lengend Snippet: The temporal dynamics of GABA A R internalization during OGD (A) Schematic depicting the signaling events involved in GABAergic synapse disassembly following OGD onset. GABA A Rs are dispersed from synaptic sites within 5 min, a process controlled by calcineurin (CaN). At 10 min, gephyrin is removed from synapses by calpain-dependent cleavage of the scaffold. Temporal kinetics of GABA A R trafficking during OGD and subsequent reperfusion remain uncharacterized. (B) Surface biotinylation of hippocampal neurons treated to an OGD time course at increasing 5 min intervals. Bar graphs show GABA A R-α1 surface levels normalized to total levels (10% input), n = 6 independent experiments. (C) Representative confocal images of dendritic segments from hippocampal neurons live-labeled for GABA A R-γ2 and treated to an OGD time-course at increasing 5 min intervals. Arrows indicate high GABA A R immunofluorescence peaks for surface (green) or internalized receptors (magenta). Filled arrowheads = surface GABA A R clusters, open arrowheads = internalized GABA A Rs. Scale = 5 μm. (D) Example line scans represent the mean fluorescence intensity for each condition across the dendritic length (from left to right) for surface (green) or internalized (magenta) receptor pools. Grayed out line in merge image depicts path of line-scan. Scale = 5 μm. (E) Quantification of the internalization index (ratio of the internalized immunofluorescence (IF)/(surface IF + internalized IF), n = 36 neurons. Values represent mean ± SEM. ∗p ≤ 0.05, ∗∗p < 0.01,∗∗∗∗p < 0.0001, one-way ANOVA, Bonferroni post hoc test (B, E).

Article Snippet: Blots were blocked in either 5% milk (TBS-T) or 5% BSA (TBS-T) for phospho-specific antibodies and probed with primary antibody overnight at 4°C with the following: GABA A R-β3 (1:5000 Neuromab 75149), GABA A R-γ2 (1:1000 Neuromab 75442), GABA A R-α1 (1:2000 Neuromab 75136), GABA A R-β3 phospho-Ser408/409 (1:1000 Rockland 612-401-D51) and GAPDH (1:40,000 GeneTex 627408).

Techniques: Labeling, Immunofluorescence, Fluorescence

Journal: iScience

Article Title: Distinct mechanisms drive sequential internalization and degradation of GABA A Rs during global ischemia and reperfusion injury

doi: 10.1016/j.isci.2023.108061

Figure Lengend Snippet: PP1α inhibition prevents OGD-induced GABA A R surface removal but not their declustering from the synapse (A) Surface biotinylation of hippocampal neurons treated to control or OGD conditions for 30 min in the presence of OkA at low (50 nM; selectively inhibits PP2A) or high (0.5 μM; inhibits PP2A and PP1α) concentrations. Quantification shows GABA A R-α1 surface levels normalized to total levels (10% input), n = 6 independent experiments. (B) Representative confocal images of dendritic segments from hippocampal neurons labeled for surface GABA A R-γ2, gephyrin and VGAT. Neurons were treated with control or OGD conditions for 30 min in the presence of OkA (at 50 nM or 0.5 μM). Arrowheads indicate VGAT positive synapses. Scale bar = 5 μm. (C) Quantification of synapse cluster area (left) and density (right) from images in (B), n = 30–36 neurons. (D) Proximity ligation assay (PLA) for gephyrin and GABA A R-α1 in GFP expressing hippocampal neurons. Representative dendritic segments show PLA signal (magenta) from neurons treated with control or OGD conditions for 30 min, in the presence of OkA (50 nM or 0.5 μM). Arrowheads indicate PLA-positive signal (representing the GABA A R-gephyrin interaction). Scale = 5 μm. (E) Quantification shows the density of the PLA signal, n = 25–32 neurons. Values represent mean ± SEM. ∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA, Bonferroni post hoc test (A, C, E).

Article Snippet: Blots were blocked in either 5% milk (TBS-T) or 5% BSA (TBS-T) for phospho-specific antibodies and probed with primary antibody overnight at 4°C with the following: GABA A R-β3 (1:5000 Neuromab 75149), GABA A R-γ2 (1:1000 Neuromab 75442), GABA A R-α1 (1:2000 Neuromab 75136), GABA A R-β3 phospho-Ser408/409 (1:1000 Rockland 612-401-D51) and GAPDH (1:40,000 GeneTex 627408).

Techniques: Inhibition, Labeling, Proximity Ligation Assay, Expressing

Journal: iScience

Article Title: Distinct mechanisms drive sequential internalization and degradation of GABA A Rs during global ischemia and reperfusion injury

doi: 10.1016/j.isci.2023.108061

Figure Lengend Snippet: PP1α-dependent dephosphorylation of the GABA A R-β3 subunit during global ischemia (A) Cartoon of GABA A R β3 subunit. The intracellular domain contains a triple arginine motif ‘RRR’ which mediates binding to AP2 and receptor endocytosis. Adjacent serine phospho-sites at 408/409 act as negative regulators of receptor endocytosis when phosphorylated. (B–C) Hippocampal neuronal lysates analyzed following control or OGD insults for 30 min ( in vitro ; B) and hippocampal crude membrane lysates from mice post sham or CA/CPR procedure ( in vivo ; C). Phosphorylated β3-S408/409 levels were normalized to total GABA A R-β3 levels, n = 5 independent cultures/animals. (D) Hippocampal neuronal lysates analyzed following a 30 min OGD insult in the presence of OkA (50 nM or 0.05 μM). Phosphorylated β3-S408/409 levels were normalized to total GABA A R-β3 levels, n = 4 independent experiments. (E) Hippocampal lysates analyzed following OGD at 3 min intervals up to a maximum of a 15 min. Phosphorylated β3-S408/409 levels were normalized to total GABA A R-β3 levels, n = 5 independent experiments. (F) Representative confocal images of dendritic segments from hippocampal neurons live-labeled for surface GABA A R-γ2 and treated to control or OGD conditions for 30 min in the presence OkA at either concentration (50 nM or 0.05 μM), or CsA. Scale bar = 5 μm. (G) Quantification of the internalization index, n = 35–36 neurons. Values represent mean ± SEM. Data in (B), (D), (E) & (F) were normalized to control conditions. ∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, Student’s t test (B, C) or one-way ANOVA, Bonferroni post hoc test (D, E, G). See also Figure S1 .

Article Snippet: Blots were blocked in either 5% milk (TBS-T) or 5% BSA (TBS-T) for phospho-specific antibodies and probed with primary antibody overnight at 4°C with the following: GABA A R-β3 (1:5000 Neuromab 75149), GABA A R-γ2 (1:1000 Neuromab 75442), GABA A R-α1 (1:2000 Neuromab 75136), GABA A R-β3 phospho-Ser408/409 (1:1000 Rockland 612-401-D51) and GAPDH (1:40,000 GeneTex 627408).

Techniques: De-Phosphorylation Assay, Binding Assay, In Vitro, Membrane, In Vivo, Labeling, Concentration Assay

Journal: iScience

Article Title: Distinct mechanisms drive sequential internalization and degradation of GABA A Rs during global ischemia and reperfusion injury

doi: 10.1016/j.isci.2023.108061

Figure Lengend Snippet: Reduced GABA A R expression during post-OGD reperfusion (A) Hippocampal neuronal lysates harvested following 30 min control or OGD treatment. GABA A R-γ2 or β3 levels were normalized to GAPDH levels, n = 5 independent experiments. (B) Hippocampal crude membrane lysates from mice immediately following sham or CA/CPR surgeries. GABA A R-γ2 or β3 levels were normalized to GAPDH levels, n = 5 independent experiments. (C) Hippocampal neuronal lysates analyzed following reperfusion up to 4 h after a 30 min OGD insult. Total GABA A R-γ2, -β3 and -α1 levels were normalized to GAPDH levels, n = 5–6 independent experiments. Colored stars compare statistical significance to control condition for each subunit. (D) Representative confocal images of dendritic segments from hippocampal neurons live-labeled for surface GABA A R-γ2 and treated to control or OGD conditions at different time-points following reperfusion injury. Scale = 5μm. Quantification of total GABA A R intensity (internal + surface), n = 33–36 neurons. (E) Hippocampal crude membrane lysates were isolated from CA1 and CA3 subregions following 4 h of reperfusion after sham or CA/CPR surgeries. GABA A R-β3 and -α1 levels were normalized to GAPDH levels, n = 6 independent experiments. Values represent mean ± SEM. ∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, Student’s t test (A, B, E), one-way ANOVA, Bonferroni post hoc test (C, D). See also Figures S2 and .

Article Snippet: Blots were blocked in either 5% milk (TBS-T) or 5% BSA (TBS-T) for phospho-specific antibodies and probed with primary antibody overnight at 4°C with the following: GABA A R-β3 (1:5000 Neuromab 75149), GABA A R-γ2 (1:1000 Neuromab 75442), GABA A R-α1 (1:2000 Neuromab 75136), GABA A R-β3 phospho-Ser408/409 (1:1000 Rockland 612-401-D51) and GAPDH (1:40,000 GeneTex 627408).

Techniques: Expressing, Membrane, Labeling, Isolation

Journal: iScience

Article Title: Distinct mechanisms drive sequential internalization and degradation of GABA A Rs during global ischemia and reperfusion injury

doi: 10.1016/j.isci.2023.108061

Figure Lengend Snippet: Internalized GABA A Rs are targeted to the lysosome for degradation during ischemic reperfusion injury (A) Representative confocal images of dendritic segments from hippocampal neurons live-labeled for surface GABA A R-γ2 and treated to control or OGD conditions at different time-points following reperfusion injury. Neurons were labeled with antibodies to lysosomal marker LAMP1 and colocalization between internalized GABA A Rs and LAMP1 was calculated by Pearson’s coefficient. Quantification shows normalized Pearson’s coefficient, n = 36 neurons per condition. Scale = 5 μm. (B) Representative confocal images of dendritic segments from hippocampal neurons live-labeled for surface GABA A R-γ2 and underwent OGD + reperfusion (2 h), with or without leupeptin. Neurons were labeled with antibodies to lysosomal marker LAMP1 and colocalization between internalized GABA A Rs and LAMP1 was calculated by Pearson’s coefficient. Quantification shows normalized Pearson’s coefficient, n = 36 neurons per condition. Scale = 5 μm. (C) Hippocampal neuronal lysates harvested following a 30 min control or OGD insult + reperfusion (2 h) in the presence of leupeptin. GABA A R-β3 levels were normalized to GAPDH levels, n = 8 independent experiments. (D) Representative confocal images of dendritic segments from hippocampal neurons live-labeled for surface GABA A R-γ2 and treated to control or OGD conditions + reperfusion in the presence of leupeptin. Scale = 5μm. Quantification of total GABA A R fluorescence (surface + internalized), n = 36 neurons. Scale = 5 μm. Values represent mean ± SEM. ∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA, Bonferroni post hoc test.

Article Snippet: Blots were blocked in either 5% milk (TBS-T) or 5% BSA (TBS-T) for phospho-specific antibodies and probed with primary antibody overnight at 4°C with the following: GABA A R-β3 (1:5000 Neuromab 75149), GABA A R-γ2 (1:1000 Neuromab 75442), GABA A R-α1 (1:2000 Neuromab 75136), GABA A R-β3 phospho-Ser408/409 (1:1000 Rockland 612-401-D51) and GAPDH (1:40,000 GeneTex 627408).

Techniques: Labeling, Marker, Fluorescence

Journal: iScience

Article Title: Distinct mechanisms drive sequential internalization and degradation of GABA A Rs during global ischemia and reperfusion injury

doi: 10.1016/j.isci.2023.108061

Figure Lengend Snippet:

Article Snippet: Blots were blocked in either 5% milk (TBS-T) or 5% BSA (TBS-T) for phospho-specific antibodies and probed with primary antibody overnight at 4°C with the following: GABA A R-β3 (1:5000 Neuromab 75149), GABA A R-γ2 (1:1000 Neuromab 75442), GABA A R-α1 (1:2000 Neuromab 75136), GABA A R-β3 phospho-Ser408/409 (1:1000 Rockland 612-401-D51) and GAPDH (1:40,000 GeneTex 627408).

Techniques: Imaging, Recombinant, SYBR Green Assay, Software

Journal: iScience

Article Title: Distinct mechanisms drive sequential internalization and degradation of GABA A Rs during global ischemia and reperfusion injury

doi: 10.1016/j.isci.2023.108061

Figure Lengend Snippet:

Article Snippet: GABA A R-β3 (WB) , NeuroMab , Cat. #75-149; RRID: AB_10673389.

Techniques: Imaging, Recombinant, Reverse Transcription, SYBR Green Assay, Software

Journal: iScience

Article Title: Distinct mechanisms drive sequential internalization and degradation of GABA A Rs during global ischemia and reperfusion injury

doi: 10.1016/j.isci.2023.108061

Figure Lengend Snippet:

Article Snippet: GABA A R-β3 phospho-Ser408/409 , Rockland , Cat. #612-401-D51; RRID: AB_11183444.

Techniques: Imaging, Recombinant, Reverse Transcription, SYBR Green Assay, Software