Journal: Cell

Article Title: Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements

doi: 10.1016/j.cell.2019.08.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MADR + AAVS1 human cell line generation AAVS1 targeting MADR vector was derived from AAVS1-targeting vector AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene 68375).

Techniques: Clone Assay, Recombinant, Lysis, Sequencing, Plasmid Preparation, Mutagenesis, Software

Journal: Frontiers in Veterinary Science

Article Title: Electrical impedance tomography in anaesthetised chickens ( Gallus domesticus )

doi: 10.3389/fvets.2024.1202931

Figure Lengend Snippet: Tomographic images of the coelom at the level of the EIT belt, in various formats. The images are in sequential order from left to right, (A–E) detailing the process of generating a chicken-specific functional EIT image. All images are aligned in the same orientation as labeled in (A) . (A) Computed tomographic (CT) image of the coelom at the level of the EIT belt. (B) Segmentation of the regions of interest (ROI), including the cardiac silhouette, air sacs, lungs, tracheal bifurcation and the body contour, using the software ITK-SNAP. Each of the segmented structures are shaded with its respective colour as indicated in the legend. (C) Model of the coelom using the mean contours of the segmentations from the CT images. The blue outlines the cardiac silhouette, the red outlines the right lung, and the green outlines the left lung. (D) The chicken-specific finite element (FE) model, created using the software Matlab. The red area represents the cardiac silhouette, and the blue represents the lungs. (E) Functional EIT image, after application of the FE model on the raw EIT data, during inspiration in one chicken. The blue represents impedance change, the gray outlines the outer contour, and the white outlines the lungs ROI. No impedance change was noted within the lungs ROI. Impedance signals were noted in the area of the air sacs and cardiac silhouette.

Article Snippet: The chickens remained in each recumbency position for 15 min: 13 min of stabilisation followed by 2 min of EIT data recording using the EIT software (BB Vet SW, SenTec, Switzerland).

Techniques: Functional Assay, Labeling, Software

Journal: Nature communications

Article Title: A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes.

doi: 10.1038/ncomms4227

Figure Lengend Snippet: Figure 2 | TWIK-1 associates with TREK-1 in heterologous systems. (a) Identification of the TREK-1 as a direct binding partner of TWIK-1 in the novel split-ubiquitin yeast two-hybrid screen. (b) Co-immunoprecipitation assay in HEK293T cells. HA-TREK-1 and FLAG-TWIK-1 or HA-TWIK-1 and FLAG-TREK-1 were transfected into HEK293T cells and cell lysates were then immunoprecipitated using anti-HA antibody. The immunoprecipitates were examined by western blotting using anti-FLAG antibody. Input represented 5% of cell lysates used in the Co-IP assay. (c) Schematic diagram depicting the BiFC assay. VN and VC are the N-terminal and C-terminal fragments of the Venus, respectively. Intense Venus signals (green colour) are examined for dimerization. TWIK-1-VC strongly interacts with VN-TREK-1; TREK-1-VC also interacts with VN-TWIK-1. In the opposite orientation (TREK-1-VN and VC-TWIK-1 or TWIK-1-VN and VC-TREK-1), Venus signals were not detected. TWIK-1 homodimer is used as a positive control, and Kir4.1 and TWIK-1 heterodimer is used as a negative control. The fluorescent signals of BiFC were detected at the plasma membrane of the cells as indicated by the yellow colour (arrow, inset magnified image). Scale bar, 20 mm.

Article Snippet: The specificities of TREK-1 antibody (Alomone Labs; #APC-047, 1:100) and TWIK-1 antibody (Santa Cruz Biotechnology; sc-11483, 1:50) were assessed with immunocytochemistry in cultured astrocytes in combination with TREK-1 shRNA or TWIK-1 shRNA.

Techniques: Binding Assay, Ubiquitin Proteomics, Yeast, Two Hybrid Screening, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Bimolecular Fluorescence Complementation Assay, Positive Control, Negative Control, Clinical Proteomics, Membrane

Journal: Nature communications

Article Title: A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes.

doi: 10.1038/ncomms4227

Figure Lengend Snippet: Figure 3 | TWIK-1 associates with TREK-1 in native astrocytes. (a) TWIK-1 co-localization with TREK-1 in astrocytes. Astrocytes were fixed and stained using specific antibodies for TWIK-1 and TREK-1. Co-localization was determined by merging individual panels. Scale bar, 20 mm. (b) Co-immunoprecipitation assay in astrocytes. Whole-cell lysates were immunoprecipitated using anti-TREK-1 antibody and then blotted using anti-TWIK-1 antibody. (c) Duolink PLA assay. Intense PLA signals were detected in Sc shRNA transfected astrocytes, but not in TWIK-1 shRNA transfected astrocytes. Scale bar, 20 mm. (d) The PLA signals were counted with Duolink Image Tool software and the average number of spots per cell is presented in the graph. Number on each bar indicates n for each condition. All values are mean±s.e.m. P-values were obtained with Student’s t-test. *Po0.05.

Article Snippet: The specificities of TREK-1 antibody (Alomone Labs; #APC-047, 1:100) and TWIK-1 antibody (Santa Cruz Biotechnology; sc-11483, 1:50) were assessed with immunocytochemistry in cultured astrocytes in combination with TREK-1 shRNA or TWIK-1 shRNA.

Techniques: Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, shRNA, Transfection, Software

Journal: Nature communications

Article Title: A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes.

doi: 10.1038/ncomms4227

Figure Lengend Snippet: Figure 5 | TWIK-1 and TREK-1 are interdependently required for cell surface expression in astrocytes. (a) Plasma membrane localization of TWIK-1 in COS-7 cells. COS-7 cells were transfected with DsRed-Mem vector and FLAG-TWIK-1 in the absence (arrow) or presence (arrowhead) of HA-TREK-1 and were labelled with anti-FLAG antibody. Scale bar, 20 mm. (b) Pearson’s correlation coefficient denoting covariance of fluorescence signals was calculated using Nikon software. The Pearson’s correlation coefficient for TWIK-1 and DsRed-Mem in the presence of TREK-1 was significantly greater than values obtained from COS-7 cells in the absence of TREK-1. Number on each bar indicates n for each condition. All values are mean±s.e.m. P-values were obtained with Student’s t-test. (c) Quantification of TWIK-1(HA) cell surface expression in the absence and presence of additional TREK-1 using anti-HA antibody. TWIK-1 cell surface expression is normalized to the average at TWIK-1-only expressing condition. Number on each bar indicates n for each condition. All values are mean±s.e.m. P-values were obtained with Student’s t-test. (d) Cell surface biotinylation assay in HEK293Tcells. When HEK293T cells were transiently transfected with GFP-TREK-1 and FLAG-TWIK-1, cell surface expression of TWIK-1 is dramatically increased. (e) Confocal immunofluorescence staining of TWIK-1 in TREK-1 shRNA transfected astrocytes. Fixed cells also stained with Alexa Fluo 647-labelled phalloidin for F-actin staining. Scale bar, 20 mm. (f) Cell surface biotinylation assay in TREK-1 shRNA-transfected astrocytes. (g) Confocal immunofluorescence staining of TREK-1 in TWIK-1 shRNA transfected astrocytes. Scale bar, 20mm. (h) Cell surface biotinylation assay in TWIK-1 shRNA-transfected astrocytes. ***Po0.001.

Article Snippet: The specificities of TREK-1 antibody (Alomone Labs; #APC-047, 1:100) and TWIK-1 antibody (Santa Cruz Biotechnology; sc-11483, 1:50) were assessed with immunocytochemistry in cultured astrocytes in combination with TREK-1 shRNA or TWIK-1 shRNA.

Techniques: Expressing, Clinical Proteomics, Membrane, Transfection, Plasmid Preparation, Software, Cell Surface Biotinylation Assay, Confocal, Staining, shRNA

Journal: Nature communications

Article Title: A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes.

doi: 10.1038/ncomms4227

Figure Lengend Snippet: Figure 6 | Astrocytes express mostly the TWIK-1/TREK-1 heterodimer and it is this channel that regulates passive conductance. (a–c) Representative whole-cell I–V curves in astrocytes over-expressing Sc shRNA (black), TWIK-1 shRNA (red), TREK-1 shRNA (blue), or TWIK-1 and TREK-1 shRNAs (green). (d) Pooled data for whole-cell current amplitudes in TWIK-1 knockdown, TREK-1 knockdown or TWIK-1 and TREK-1 double knockdown astrocytes. Number on each bar indicates n for each condition. All values are mean±s.e.m. (e,f) Normalized current density. Data are mean±s.e.m. (g) DIC (upper) and fluorescence (lower) image of hippocampal astrocyte infected with mCherry-TREK-1 shRNA lentivirus in the CA1 striatum radiatum. Scale bar, 20 mM. (h) The whole-cell currents from hippocampal astrocytes infected with Sc (left) or TWIK-1 (middle) or TREK-1 (right) shRNA. Command voltage pulse protocol: voltages were stepped from 160 mV to þ 40 mV in increments of 20 mV from a holding potential of 60 mV. Dotted line indicates 0 pA. (i) The current–voltage relationship (I–V) from 160 mV to þ 40 mV. (j) The bar graph of current at 160 mV and þ 40 mV. Data are mean±s.e.m. P-values were obtained with Student’s t-test. **Po0.01. Numbers of tested slices from at least two independent mice are indicated on each bar. (k) The bar graph representing reversal potential of respective conditions. Data are mean±s.e.m. P-values were obtained with Student’s t-test. *Po0.05, **Po0.01.

Article Snippet: The specificities of TREK-1 antibody (Alomone Labs; #APC-047, 1:100) and TWIK-1 antibody (Santa Cruz Biotechnology; sc-11483, 1:50) were assessed with immunocytochemistry in cultured astrocytes in combination with TREK-1 shRNA or TWIK-1 shRNA.

Techniques: Expressing, shRNA, Knockdown, Infection

Journal: Nature communications

Article Title: A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes.

doi: 10.1038/ncomms4227

Figure Lengend Snippet: Figure 7 | TWIK-1/TREK-1 heterodimer releases glutamate upon CB1 activation in astrocytes. (a–c) Average I–V curves for TWIK-1 or TREK-1 shRNA- sensitive currents in brain slices, cultured astrocytes and COS-7 cells expressing both TWIK-1 and TREK-1. (d) Representative traces for TFLLR-induced fast and slow glutamate release from astrocytes expressing Sc shRNA (upper panel) and TWIK-1 shRNA (lower panel). The diamonds indicate pressure application of TFLLR (100 ms, 0.1 s). (e) Summary bar graph for % of full activation of results from astrocytes expressing Sc shRNA, TREK-1 shRNA and TWIK-1 shRNA. All values are mean±s.e.m. P-values were obtained with Student’s t-test. (f) Representative traces for ACEA-induced fast glutamate release from cultured astrocytes expressing Sc shRNA, TWIK-1 shRNA, TWIK-1 plus TWIK-1 shRNA-insensitive form (rescue), and TWIK-1 shRNA plus TWIK-1 shRNA-insensitive form C93S mutant (rescue control). (g) Summary bar graph for % of full activation of results from astrocytes expressing TWIK-1 plus TWIK-1 shRNA-insensitive form (rescue) and TWIK-1 shRNA plus TWIK-1 shRNA-insensitive form C93S mutant (rescue control). All values are mean±s.e.m. P-values were obtained with Student’s t-test. (h) Schematic diagrams for passive conductance (left panel) and Gbg-dependent glutamate release (right panel). *Po0.05, ***Po0.001.

Article Snippet: The specificities of TREK-1 antibody (Alomone Labs; #APC-047, 1:100) and TWIK-1 antibody (Santa Cruz Biotechnology; sc-11483, 1:50) were assessed with immunocytochemistry in cultured astrocytes in combination with TREK-1 shRNA or TWIK-1 shRNA.

Techniques: Activation Assay, shRNA, Cell Culture, Expressing, Mutagenesis, Control

Journal: Cell reports

Article Title: TALK-1-mediated alterations of β-cell mitochondrial function and insulin secretion impair glucose homeostasis on a diabetogenic diet

doi: 10.1016/j.celrep.2024.113673

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rehydrated 5-μm sections were stained with primary antibodies against insulin (dilution 1:500; Dako, Santa Clara, CA, USA), somatostatin (dilution 1:250; sc-55565, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and TALK-1 (dilution 1:175; APC-170, Alomone Labs, Jerusalem, Israel; or dilution 1:175; Prestige Antibodies Novus Biologicals #NBP1–83071) followed by secondary antibodies (dilution 1:500; anti-guinea pig Alexa Fluor 647, dilution 1:500; anti-mouse Alexa Fluor 488, and dilution 1:500; anti-rabbit Alexa Fluor 546). β-cell proliferation in response to HFD feeding was quantified by immunostaining for Ki-67 (dilution 1:300; ab15580, Abcam) and utilizing CytoNuclear FL v1.2 algorithm macros generated in HALO software (Indica Labs) to count insulin and Ki-67 double-positive cells.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software