Journal: Cell reports

Article Title: Activity- and sleep-dependent regulation of tonic inhibition by Shisa7

doi: 10.1016/j.celrep.2021.108899

Figure Lengend Snippet: (A) Shisa7 interacted with α5 in HEK293T cells. HEK293T cells were transfected with Flag-Shisa7, α5-GFP, or both Flag-Shisa7 and α5-GFP. Cell lysates were subjected to immunoprecipitation and immunoblotting assays (n = 3 independent experiments). (B) α5, but not α4 or δ, subunit was co-immunoprecipitated with Shisa7 in detergent-solubilized mouse hippocampal lysates. Asterisk indicates the Shisa7 band (n = 3 independent experiments). (C) Co-expression of α5β3γ2 with Shisa7-IRES-GFP (Shisa7/GFP), but not GFP, increased surface levels, total levels, and surface to total ratio of α5 expression (GFP, n = 23; Shisa7/GFP, n = 27; surface α5: t test, p < 0.0001; total α5: Mann-Whitney U test, p = 0.0298; surface α5/ total α5: t test, p = 0.0009). (D) GABA-evoked α5β3γ2-mediated whole-cell currents were significantly increased in HEK293T cells co-expressing Shisa7/GFP. (GFP, n = 17; Shisa7/GFP, n = 14, t test, p = 0.0001). (E) Immunostaining (left) and summary graphs (right) showing surface and total α5 expression were reduced in Shisa7 KO neurons (WT, n = 22; KO, n = 20; surface α5: Mann-Whitney U test, p < 0.0001; total α5: Mann-Whitney U test, p < 0.0001; surface α5/total α5: Mann-Whitney U test, p < 0.0001). (F) Representative images (left) of SEP-α5 fluorescence and the regions of the neuronal dendrites used for the fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments. Repetitive photobleaching (FLIP, white box) occurred at regions bilateral to the central FRAP region (red box). Each column (right) represents before (pre), immediately after (t = 0′), and at 2 min (t = 2′) and 5 min (t = 5′) after photobleaching in each condition. (G) Normalized fluorescence recovery curves showing significantly less newly inserted α5 on the cell surface in Shisa7 KO neurons than in WT neurons 5 min after photobleaching, indicating that α5-GABA A Rs exocytosis was impaired inShisa7 KO neurons (WT, n = 6; KO, n = 8, two-way ANOVA with Tukey’s multiple comparison test. t = 5′: p < 0.0001; t = 5.5′: p = 0.0002; t = 6′: p < 0.0001; t = 6.5′: p = 0.0002). *p < 0.05, ***p < 0.001, and ****p < 0.0001. All data are presented as mean ± SEM. See also .

Article Snippet: Rabbit Polyclonal Anti-GABA(A) α4 Receptor , Alomone Labs , Cat # AGA-008; RRID: AB_10917596.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, MANN-WHITNEY, Immunostaining, Fluorescence

Journal: Cell reports

Article Title: Activity- and sleep-dependent regulation of tonic inhibition by Shisa7

doi: 10.1016/j.celrep.2021.108899

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Polyclonal Anti-GABA(A) α4 Receptor , Alomone Labs , Cat # AGA-008; RRID: AB_10917596.

Techniques: Recombinant, Transfection, Protease Inhibitor, Knock-Out, Software

Journal: The European Journal of Neuroscience

Article Title: Tonic GABAergic inhibition, via GABA A receptors containing αβƐ subunits, regulates excitability of ventral tegmental area dopamine neurons

doi: 10.1111/ejn.15133

Figure Lengend Snippet: Primary antibodies

Article Snippet: GABA A α4 , RbP , 1:500 , Alomone Labs , AGA‐008 , AB_10917596.

Techniques:

Journal: bioRxiv

Article Title: Brain neurosteroids are natural anxiolytics targeting α2 subunit γ-aminobutyric acid type-A receptors

doi: 10.1101/462457

Figure Lengend Snippet: (a) Southern blot screen of ES cell lines: the Q241M mutation silently introduces a novel restriction site – allowing a screen for restriction fragment length polymorphism. Lanes 1 - 5, heterozygous recombinants; lanes 6 - 7, wild-type littermate controls. (b) Verifying the point mutation by DNA sequencing. Exon 8 was PCR-amplified from mouse genomic DNA of wild-type (α2 Q/Q : left), α2 Q/M (middle) and α2 M/M (right) animals, then sequenced. Sequence data around the point mutation are shown. (c,d) GABA A R α1-α4 subunit expression levels were determined by Western blotting total protein isolated from the cortex ( c ) and hippocampus ( d ). Left panels, quantitation of expression in α2 Q/M (grey) and α2 M/M (white) relative to wild-type (α2 Q/Q , black) brain samples. Right panels, representative Western blots from cortex and hippocampus for wild-type (WT), α2 Q/M and α2 M/M mice. Images show blots between 45 and 66 kDa markers. Loading controls with tubulin are omitted for clarity. Data are mean ± sem, one-way ANOVA, n = 6-9. There are no statistically significant differences between groups.

Article Snippet: For immunohistochemistry, the following primary antibodies were used: rabbit anti-α1-GABA A R (1:20000, from Jean-Marc Fritschy, University of Zurich), guinea-pig anti-α2-GABA A R (1:1000, from Jean-Marc Fritschy), rabbit anti-α3-GABA A R (1:1000, Alomone Labs), rabbit anti-α4-GABA A R (1:500, Werner Sieghart), rabbit anti-α5-GABA A R (1:500, Werner Sieghart).

Techniques: Southern Blot, Mutagenesis, DNA Sequencing, Amplification, Sequencing, Expressing, Western Blot, Isolation, Quantitation Assay