Journal: bioRxiv

Article Title: One-shot design elevates functional expression levels of a voltage-gated potassium channel

doi: 10.1101/2022.12.28.522065

Figure Lengend Snippet: (a) Currents of the paddle chimera (PC) and designs are plotted against varying test voltages. Each curve represents recordings from n ⩾8 oocytes. Error bars represent standard deviations. (b) Average currents (paddle chimera: 0.91±0.19 μA, D1: 4.65±1.52 μA, D2: 12.50±1.63 μA). (c) Western blot results reporting total membrane expression. The blots were labeled with anti-FLAG (top) or with anti-GIRK2 (as control; bottom) antibodies. (d) Normalized blot intensities. The experiment was performed in triplicate. Error bars represent the standard deviation. (e) Normalized tail currents were recorded at -50mV as a function of the test pulse voltage (see inset). Measured tail current amplitudes were fitted with a single-component Boltzmann equation (solid lines), from which V 1/2 of activation was inferred. (f) Inferred V 1/2 values (paddle chimera: 16.10±5.39 mV, D1: -8.33±3.49 mV, and, D2: -16.61±2.75 mV).

Article Snippet: For the controls, anti-GIRK2 (Alomone lab, catalog No. APC-006) and HRP conjugated anti-rabbit secondary antibodies were added to label intrinsic membrane proteins expressed in Xenopus oocytes.

Techniques: Western Blot, Expressing, Labeling, Standard Deviation, Activation Assay

Journal: Neural Development

Article Title: Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei

doi: 10.1186/1749-8104-6-29

Figure Lengend Snippet: Cells labeled with Shh-GIFM at different time points have a changing potential to contribute to subpopulations of DA neurons . (A-H) Immunofluorescent staining for β-gal-positive fate-mapped cells (Shh-GIFM at E8.5 or E11.5) and DA neurons (TH) on coronal sections of the ventral midbrain (P21 to P30). The areas shown in (A-D) are indicated in (E-H). Arrows indicate double-labeled cells; arrowheads indicate β-gal-positive cells with astrocytic morphology. (E'-H') Representative schematics of the immunostained sections showing the distribution of TH-positive fate-mapped cells (red dots) and of fate-mapped cells with astrocytic morphology (yellow crosses). Rostral, Bregma -2.92; caudal, Bregma -3.40 . If, interfascicular nucleus; Pn, paranigral nucleus; Snc, substantia nigra pars compacta; Snl, substantia nigra lateralis; Vta, VTA. Fate-mapped cells outside these areas are not represented. Cells with astrocytic morphology are not present with TM8.5. Scale bars: (A-D) 40 μm; (E-H) 200 μm. (I) Relative contribution of cells marked with Shh-GIFM between E8.5 and E11.5 to the SN (Snl + Snc), dorsal-lateral VTA (Vta) and ventral-medial VTA (Pn + If); see schematic in (L). For each animal (n ≥ 3), TH-positive fate-mapped cells were counted in the three indicated areas and normalized for the combined number of overlapping cells counted in these areas (in percent). Error bars indicate standard deviation. Significance (* P < 0.05; ** P < 0.01; *** P < 0.001) was determined by ANOVA and LSD post-hoc analysis. (J) Distribution of Calbindin- and Girk2-positive cells. (K) Relative contribution of fate-mapped cells to Calbindin (VTA) versus Girk2 (SN) positive cells. Calbindin- or Girk2-positive fate-mapped cells were counted in three different rostral-caudal midbrain areas (n ≥ 3). The ratio of Calbindin-positive fate-mapped cells to Girk2-positive fate-mapped cells was determined. Significance (*** P < 0.001) was determined by Student's t -test. (L) Schematic showing the SN, dlVTA and vmVTA. (M) Fate mapping strategy.

Article Snippet: Primary antibodies: goat anti-β-galactosidase (β-gal; 1:2,000, AbD Serotec, Oxford, UK), rabbit or rat anti-GFP (1:400, Invitrogen, Carlsbad, CA, USA or 1:2,000, Nacalai, Kyoto, Japan) rabbit or mouse anti-tyrosine hydroxylase (TH; 1:500, Millipore, Billerica, MA, USA), rabbit anti-Calbindin (1:5,000, Swant, Marly, Switzerland), rabbit anti-Lmx1a (1:2,000, Millipore), mouse anti-Pou4f1 (1:100, Santa Cruz Antibodies, Santa Cruz, CA, USA), rabbit anti-glial fibrillary acidic protein (GFAP; 1:500, Millipore), mouse anti-glutamine synthetase (1:500, Millipore) and rabbit anti-Girk2 (1:100, Alomone Labs, Jerusalem, Israel).

Techniques: Labeling, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Spatial Memory Training Counteracts Hippocampal GIRK Channel Decrease in the Transgenic APP Sw,Ind J9 Alzheimer’s Disease Mouse Model

doi: 10.3390/ijms232113444

Figure Lengend Snippet: Effect of genotype and age on hippocampal levels of GIRK channel subunits and modulator. Protein levels of GIRK channel subunits (1–4) and modulator (RGS7) in the hippocampus of non-transgenic (WT) and APP Sw,Ind J9 mice at 6 and 12–18 months of age. ( A ) GIRK1, ( B ) GIRK2, ( C ) GIRK3, ( D ) GIRK4 and ( E ) RGS7. Data is expressed as percentage relative to 6-month-old WT mice (100%). ** p < 0.01, *** p < 0.001 vs. WT 6-month-old; # p < 0.05 vs. APP Sw,Ind J9 6-month-old. m, months; βAct, β actin.

Article Snippet: GIRK2 , APC-006 , Alomone, Jerusalem, Israel , Rabbit , 1/500.

Techniques: Transgenic Assay

Journal: International Journal of Molecular Sciences

Article Title: Spatial Memory Training Counteracts Hippocampal GIRK Channel Decrease in the Transgenic APP Sw,Ind J9 Alzheimer’s Disease Mouse Model

doi: 10.3390/ijms232113444

Figure Lengend Snippet: Effect of genotype and spatial memory training on hippocampal levels of GIRK channel subunits and modulator. Protein levels of GIRK channel subunits (1–4) and modulator (RGS7) in the hippocampus of naïve and memory-trained non transgenic (WT) and APP Sw,Ind J9 mice at 6 months of age. ( A ) GIRK1, ( B ) GIRK2, ( C ) GIRK3, ( D ) GIRK4 and ( E ) RGS7. Data is expressed as percentage relative to untrained naïve WT mice (100%). ** p < 0.01 vs. naïve WT. βAct, β actin.

Article Snippet: GIRK2 , APC-006 , Alomone, Jerusalem, Israel , Rabbit , 1/500.

Techniques: Transgenic Assay

Journal: International Journal of Molecular Sciences

Article Title: Spatial Memory Training Counteracts Hippocampal GIRK Channel Decrease in the Transgenic APP Sw,Ind J9 Alzheimer’s Disease Mouse Model

doi: 10.3390/ijms232113444

Figure Lengend Snippet: Characteristics of primary and secondary antibodies used to measure protein expression by Western Blot.

Article Snippet: GIRK2 , APC-006 , Alomone, Jerusalem, Israel , Rabbit , 1/500.

Techniques: Expressing, Western Blot

Journal: Brain Pathology

Article Title: Targeting the interaction of GABA B receptors with CaMKII with an interfering peptide restores receptor expression after cerebral ischemia and inhibits progressive neuronal death in mouse brain cells and slices

doi: 10.1111/bpa.13099

Figure Lengend Snippet: R1‐Pep treatment reduced aberrant expression of K ir 3.2 channels after glutamate stress. Glutamate‐stressed neurons were treated for 16 h with R1‐Pep (10 μg/ml) or Ctrl‐Pep (10 μg/ml) and then stained with K ir 3.2 antibodies. (A) Immunofluorescence staining: top, representative images (scale bar: 5 μm); bottom, quantification of fluorescence intensities. N = 30 neurons per condition from two independent experiments. Signals were normalized to no Pep control. Two‐way ANOVA with Tukey's multiple comparison test (ns, p > 0.05; ****, p < 0.0001). (B) Western blotting: top panel, staining for K ir 3.2 channels; bottom panel, staining for total protein used for normalization. Control, untreated cultures; Glu, glutamate‐stressed cultures; Glu + R1‐Pep, glutamate‐stressed cultures treated with R1‐Pep; Glu + R1‐Pep + CGP, glutamate‐stressed cultures treated with R1‐Pep and the GABA B receptor antagonist CGP 56999. Signals were normalized to control. N = 11 cultures per condition from four independent neuron preparations. One‐way ANOVA with Tukey's multiple comparison test (ns, p > 0.05; *, p < 0.05; **, p < 0.01 ***, p < 0.0005).

Article Snippet: Mouse Akt (1:250 for immunofluorescence [IF], Cell Signaling Technology #2920), rabbit pAkt‐S473 (1:1000 for IF, Cell Signaling Technology #4060), rabbit CaMKII (1:1000 for IF, 1:100 for PLA; Abcam #ab52476), mouse CaMKIIβ (1:1500 for Western blotting [WB], Thermo Fisher Scientific #13‐9800), rabbit phospho‐CaMKII(Thr286) (1:500 for WB, Cell Signaling Technology #12716), rabbit Ca V 2.2 (1:1000 for IF and 1:200 for Western blotting; Alomone Labs #ACC‐002), mouse GABA B1 (1:250 for IF and Western blotting, 1:100 for PLA; Abcam #ab55051), rabbit GABA B1b directed against the N‐terminus of GABA B1b (affinity‐purified, 1:100 for IF; custom made by GenScript) [ ], rabbit GABA B2 directed against the N‐terminus of GABA B2 (affinity‐purified, used for cell surface staining, 1:25 for IF; custom made by GenScript) [ ], rabbit GABA B2 (1:500 for IF, 1:100 for PLA, 1:800 for Western blotting; Abcam #ab75838), mouse GFP antibody (for immunoprecipitation, TaKaRa #632381), rabbit K ir 3.2 (1:250 for IF and Western blotting, Alomone Labs #APC‐006), mouse GSK3β (1:250 for IF, Abcam #ab93926), rabbit pGSK3β‐S9 (1:1500 for IF, Abcam #ab131097), rabbit pGSK3β‐Y216 (1:1500 for IF, Abcam #ab75745), rabbit ubiquitin K63‐specific (clone Apu3, 1:50 for PLA; Millipore #05‐1308), rabbit NeuN (1:400 for IF; Millipore #ABN78), mouse phospho‐serine (1:150 for PLA, Sigma‐Aldrich #P5747), Alexa Fluor 647 and 488‐conjugated streptavidin (1:100; Jackson ImmunoResearch Laboratories #016‐600‐084 and #016‐540‐084).

Techniques: Expressing, Staining, Immunofluorescence, Fluorescence, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Targeting the Interaction of GABA B Receptors With CHOP After an Ischemic Insult Restores Receptor Expression and Inhibits Progressive Neuronal Death

doi: 10.3389/fphar.2022.870861

Figure Lengend Snippet: R2-Pep normalize OGD-mediated dysregulation of K ir 3.2 channels. Cultures were stressed for 1 h with OGD and immediately treated with R2-Pep, Ctrl-Pep or remained untreated. After 16 h neurons were immunostained for K ir 3.2 channel expression. Top: representative images (scale bar: 5 μm). Bottom: quantification of fluorescence intensities. The fluorescence intensity of the untreated neurons served as control. The data represent the mean ± S.E.M. of 37–38 neurons from two independent preparations. ns: p > 0.05; ****, p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: For monitoring K ir 3.2 channel expression, the coverslips were washed with PBS, fixed for 30 min in 4% PFA, followed by permeabilization with 0.2% Triton X-100 in PBS for 12 min and incubation with anti-K ir 3.2 antibody (1:250, Cat No. APC-006, Alomone Labs).

Techniques: Expressing, Fluorescence