Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 cleaves contactin-2 close to its GPI domain. A) Schematic representation of partial amino acid sequence of contactin-2 indicating putative BACE1 cleavage site. B) Graphic representation of GPI-anchored and soluble contactin-2 proteins that were used in this study. C) Lentiviral-mediated overexpression of BACE1 increased contactin-2 and sAPP shedding in CHO cells stably expressing GPI-anchored human contactin-2 (CNTN2). (D) Release of soluble contactin-2 (sCNTN2) was not changed by BACE1 overexpression. E and F) Quantitative analysis of the secreted contactin-2 levels in C) and D) , respectively (Student t test; *, p < 0.05; n = 3 for each condition).

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Sequencing, Over Expression, Stable Transfection, Expressing

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 inhibition reduces contactin-2 shedding. A) Western blot showing a sharp decrease in contactin-2 levels in CHO(CNTN2) cells treated with BACE1 Inhibitor IV. B) Quantitative analysis of the secreted contactin-2 levels in A) (Student t test; *, p < 0.05; n = 3 for each condition). C) Confocal microscopy images of cell surface contactin-2 levels in CHO cells expressing CNTN2. BACE1 Inhibitor IV induced a strong increase in contactin-2 surface levels (green). Nuclear staining with DAPI is shown in blue. Images are taken at identical settings.

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Inhibition, Western Blot, Confocal Microscopy, Expressing, Staining

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 inhibition also decreases release of endogenous neuronal contactin-2. A) Western blot analysis of mouse primary neuronal cultures treated with 1 μM BACE 1 Inhibitor IV showed a significant decrease in contactin-2 and sAPP levels in the conditioned media. B) Quantitative analysis of secreted contactin-2 levels in A) (Student t test; **, p < 0.01; n = 3 for each condition).

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Inhibition, Western Blot

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: Site-directed mutagenesis of the putative BACE1 cleavage site specifically blocks contactin-2 shedding. A) Partial amino acid sequence of human contactin-2 indicating the putative BACE1 cleavage site. CNTN2-MM1008AA mutations were introduced to block BACE1 cleavage. B) Western blot analysis showing a dramatic reduction in contactin-2 release in the conditioned media of the cells expressing the BACE1-cleavage site mutant contactin-2 (CNTN2-MM1008AA). C) Quantitative analysis of secreted contactin-2 levels in B) .

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Mutagenesis, Sequencing, Blocking Assay, Western Blot, Expressing

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: BACE1 regulates surface levels of contactin-2 in mouse primary neuronal cultures. A) Western blot analysis showed that surface levels of contactin-2 were largely decreased by BACE1 overexpression in mouse primary neuronal cultures. B) Quantitative analysis of surface contactin-2 and the control N-cadherin levels in A) (Student t test; ***, p < 0.005; n = 3 for each condition). C) Western blot analysis showed that surface levels of contactin-2 were largely increased by BACE1 Inhibitor IV treatments. D) Quantitative analysis of surface contactin-2 and the control NrCAM levels in C) . (Student t test; ***, p < 0.005; n = 3 for each condition).

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Western Blot, Over Expression

Journal: Molecular Neurodegeneration

Article Title: BACE1 activity regulates cell surface contactin-2 levels

doi: 10.1186/1750-1326-9-4

Figure Lengend Snippet: Contactin-2 levels are decreased in AD brain samples inversely correlating with BACE1 levels or β-amyloid plaques. A) Western blot analysis showed that contactin-2 levels were decreased in AD brain samples as compared to age-matched controls. B) Quantitative analysis of contactin-2 levels in A) (**, p < 0.01, Student’s t test; n = 9 for AD, 8 for non-AD). Contactin-2 levels were inversely correlated with BACE1 levels ( C , p = 0.032, Person’s correlation test; n = 17) or amyloid plaque densities ( D , p = 0.0064) in these samples.

Article Snippet: Anti-human contactin-2 (MAB-1714) and anti-mouse contactin-2 (AF4439) antibodies were purchased from R&D Systems (Minneapolis, USA).

Techniques: Western Blot

Journal: eLife

Article Title: p75NTR prevents the onset of cerebellar granule cell migration via RhoA activation

doi: 10.7554/eLife.79934

Figure Lengend Snippet: ( A ) Mouse developmental expression of p75NTR (green), Pax6 (magenta), DCX (white), and Dapi (blue) at the indicated ages. Note the high level of p75NTR in the meninges as well as the developing granule cell progenitors. Yellow arrows indicate the M – meninges, RL – rhombic lip, EGL – external granule layer. ( B ) High magnification of the insets showed in A. Cells expressing p75NTR (arrows), migrating cells negative for p75NTR (arrowheads). ( C ) Expression of p75NTR (green), Ki67 (magenta), and Dapi (blue) in the cerebellum of P7 mouse pups. ( D ) Immunohistochemistry of the expression of p75NTR (green), TAG1 (magenta), and Dapi (blue) in the cerebellum of P7 mouse pups. ( E ) Immunohistochemistry of the expression of p75NTR (green), DCX (magenta), and Dapi (blue) in the cerebellum of P7 mouse pups. ( F ) High magnification of the inset showed in E. The tissue shown in all the figures were obtained from mice.

Article Snippet: Antibodies used were: Ki67 (Abcam 15580, RRID: AB_443209 , 1:500), anti-p75 (R&D AF367, RRID: AB_2152638 , 1:500), anti-p75 (Millipore MAB365, RRID: AB_2152788 , 1:1000), anti-PAX-6 (BD Bioscience, RRID: AB_397991 , 1/500), anti-DCX (Abcam 18723, RRID: AB_732011 , 1/500), and anti-TAG1 (R&D Systems AF1714, RRID: AB_2245173 , 1/500).

Techniques: Expressing, Immunohistochemistry

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: Parallel fiber growth was enhanced in both Ythdf1 and Ythdf2 cKO mice. A,B) Representative images of A) YTHDF1 and B) YTHDF2 immunostaining in P15 cerebellum of A) Ythdf1 and B) Ythdf2 cKO, respectively. YTHDF1 or YTHDF2 was successfully eliminated in GCs while their expression in PCs was not affected. Scale bars represent 500 µm. C,D) Lengths of parallel fibers labeled by DiI were significantly increased in Ythdf1 and Ythdf2 cKO mice. The white arrowheads indicate the terminals of DiI‐labeled PFs. Scale bars represent 100 µm. E,F) Quantification of parallel fiber (PF) lengths in (C) and (D). Data are expressed as box and whisker plots. In (E), **** p = 1.38E‐05; for Ythdf1 fl/fl mice, n = 36 confocal fields from 11 pups, for Ythdf1 cKO mice, n = 42 confocal fields from 11 pups. In (F), **** p = 2.29E‐05; for Ythdf2 fl/fl mice, n = 46 confocal fields from 13 pups, for Ythdf2 cKO mice, n = 43 confocal fields from 12 pups. All by unpaired Student's t test. G,H) Significantly higher Tag1 IF in the deep layer of cerebellar EGL of Ythdf1 and Ythdf2 cKO mice was detected. Scale bars represent 40 µm. I,J) Quantification of Tag1 IF intensity signals in (G) and (H). Data are expressed as box and whisker plots. In (I), **** p = 2.80E‐06; n = 18 confocal fields for Ythdf1 fl/fl mice, n = 15 confocal fields for Ythdf1 cKO mice. In (J), **** p = 2.02E‐12; n = 26 confocal fields for Ythdf2 fl/fl mice, n = 28 confocal fields for Ythdf2 cKO mice. All by unpaired Student's t test.

Article Snippet: Sources and dilutions of antibodies used in WB are as follows: YTHDF1 (17479‐1‐AP, Proteintech) 1:2500; YTHDF2 (24744‐1‐AP, Proteintech) 1:2500; Dvl1 (27384‐1‐AP, Proteintech) 1:1000; Wnt5a (55184‐1‐AP, Proteintech) 1:1000; HA (ab18181, Abcam) 1:2000; Tau1 (MAB3420, Millipore) 1:1000; Tag1 (AF1714, R&D Systems) 1:2500; GluR δ 2(sc‐26118, Santa Cruz Biotechnology) 1:1000; Nrxn1 (ab77596, Abcam) 1:1000; GAPDH (10494‐1‐AP, Proteintech) 1:1000; β ‐actin (ab6276, Abcam) 1:30 000; β ‐actin (AC004, Abclonal) 1:30 000; Donkey anti‐goat IgG H&L (HRP) (ab97110, Abcam) 1:2500; Donkey anti‐mouse IgG H&L (HRP) (ab97030, Abcam) 1:2500; Donkey anti‐rabbit IgG H&L (HRP) (ab16284, Abcam) 1:2500; VHH anti‐mouse IgG secondary antibody (HRP) (KTSM1321, AlpaLife) 1:5000; VHH anti‐rabbit IgG secondary antibody (HRP) (KTSM1322, AlpaLife) 1:5000.

Techniques: Immunostaining, Expressing, Labeling, Whisker Assay

Journal: Nature Communications

Article Title: Nkx2-5 defines distinct scaffold and recruitment phases during formation of the murine cardiac Purkinje fiber network

doi: 10.1038/s41467-020-19150-9

Figure Lengend Snippet: a Scheme of the strategy used for clonal genetic tracing of Cx40 + cells. R26R-Confetti multicolor reporter mice are crossed with tamoxifen-inducible Cx40-CreERT2 mice. Four alternative recombination (nGFP, YFP, RFP, and mCFP) are possible. b Low doses of 4-hydroxytamoxifen (4’OH-Tam) are injected at different time points of development to induce low-frequency recombination. Analyses of independent unicolor clones are made at P21. c 3D reconstructions of clones imaged at the subendocardial surface of the left ventricle. Whole-mount immunostaining for Contactin-2 (CNTN2) is used to distinguish conductive, mixed and non-conductive clones. Arrowheads indicate cells positive for CNTN2. Schemes illustrate the lineage origin of each clone. d Confocal images of whole-mount CNTN2-immunostaining of 3-week-old Cx40-CreERT2::R26R-Confetti opened-left ventricle induced at E9.5 from control (Ctrl) and Nkx2-5 +/− hearts. Conductive clones are indicated by arrowheads. Scale bars = 100 µm. e Percentages of conductive, mixed and non conductive clones induced at E9.5 were quantified in P21 control (Ctrl) and Nkx2-5 +/ − left ventricles. f Clone frequency evolution upon time-course inductions quantified in P21 control ( Ctrl) and Nkx2-5 +/− mice. The progressive decrease of mixed clones over time illustrates the kinetics of Cx40 + trabecular progenitors segregation. Dashed lines: non-linear regression curves with R square values. g Graphs of the percentage of labeled adult PFs according to Cx40 + lineage traced by Tam injections at E9.5, E14.5, or E18.5. Box plots show the median, the 25th and 75th percentile, and the whiskers denote the minimum and maximum values, respectively. Quantifications were performed on Cx40-CreERT2::R26R-YFP control (ctrl) or Nkx2-5 +/ − heart transverse sections. Mean of 3 sections/heart (Tam E14.5; Tam E18.5); 4 sections/heart (TamE9.5); ( n = 3 TamE9.5 Ctrl, TamE18.5 Ctrl and Nkx2-5 +/ − ; n = 4 TamE9.5 Nkx2-5 +/ − , TamE14.5 Ctrl and Nkx2-5 +/ − ). P values are derived ordinary one-way ANOVA test (Tukey test), * P < 0.05.

Article Snippet: Antibodies used in this study are specific to sheep GFP (1:500; AbD Serotec, Bio-Rad, Hercules, CA, USA), chick GFP (1:500; 2010, Aves), and goat Contactin-2 (1:100; AF1714 R&D Systems, Minneapolis, MN, USA), Donkey anti-goat-488 (1:500; A11055, Life Technologies), Donkey anti-goat-647 (1:250; A21447, Life technologies), Donkey anti-chick-488 (1:500; 703 545 155, Interchim), WGA-555 (1:500; Wheat germ agglutinin, Clinisciences).

Techniques: Injection, Clone Assay, Immunostaining, Labeling, Derivative Assay

Journal: Nature Communications

Article Title: Nkx2-5 defines distinct scaffold and recruitment phases during formation of the murine cardiac Purkinje fiber network

doi: 10.1038/s41467-020-19150-9

Figure Lengend Snippet: a Scheme illustrating the genetic clonal tracing strategy using R26R-Confetti multicolor reporter mouse line crossed with tamoxifen-inducible Sma-CreERT2 mice. b Time course of 4-hydroxytamoxifen (4’OH-Tam) injections and analyses of independent unicolor clones represented on a time scale. Low doses of 4′OH induce low-frequency recombination. c 3D reconstructions of conductive and mixed clones induced at E7.75 and observed within Sma-CreERT2::R26R-Confetti opened-left ventricles at P21 of control (Ctrl) and Nkx2-5 +/ − left ventricle. Whole-mount immunostaining for Contactin-2 (CNTN2) is used to distinguish conductive cells (CNTN2+) as indicate by arrowheads. d Conductive versus mixed clones frequency evolution upon time-course inductions quantified in P21 control ( Ctrl ) and Nkx2-5 +/− left ventricle. The progressive decrease of mixed clones frequency over time illustrates the kinetics of Sma + cardiac progenitors segregation. e Diagram of the relative frequency of mixed clone sizes induced at E8.5 in P21 control and Nkx2-5 +/− left ventricles. P value is derived from non-parametric Mann–Whitney t- test, ** p < 0.01.

Article Snippet: Antibodies used in this study are specific to sheep GFP (1:500; AbD Serotec, Bio-Rad, Hercules, CA, USA), chick GFP (1:500; 2010, Aves), and goat Contactin-2 (1:100; AF1714 R&D Systems, Minneapolis, MN, USA), Donkey anti-goat-488 (1:500; A11055, Life Technologies), Donkey anti-goat-647 (1:250; A21447, Life technologies), Donkey anti-chick-488 (1:500; 703 545 155, Interchim), WGA-555 (1:500; Wheat germ agglutinin, Clinisciences).

Techniques: Clone Assay, Immunostaining, Derivative Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Nkx2-5 defines distinct scaffold and recruitment phases during formation of the murine cardiac Purkinje fiber network

doi: 10.1038/s41467-020-19150-9

Figure Lengend Snippet: a Scheme illustrating the genetic tracing strategy using R26R-Confetti multicolor reporter mouse line crossed with non-inducible Mesp1-Cre mice. b Recombination of the confetti allele occurs during the short time-window of Mesp1 expression, between E6.25 and E7.75. Mosaic tracing analysis in control and Nkx2-5 +/ − mice is performed at P21. c Whole-mount fluorescence views of Mesp1-Cre::R26R-Confetti opened-left ventricles at P21. Immunostaining for Contactin-2 (CNTN2) is used to label the mature PF network. Small panels (1–4) show high magnification confocal images of different PF network regions. Scale bars = 1 mm and 100 µm in insets. d Small panels (1–4) show high magnification confocal images of the PF network. Panels (1′–4′) are image reconstructions in which conductive only confetti+ cells are manually drawn. Non-conductive confetti+ cells are not represented. In control hearts, conductive confetti+ cells form either small or large monochromatic clusters. Large clusters (arrows) contribute to complex parts of the PF network. In Nkx2-5 +/ − hearts, only small clusters contribute to the hypoplastic network. Scale bars = 1 mm and 200 µm in insets. e and f Dot plots of individual Mesp1 + c onfetti colors showing percentage contribution to the working myocardium versus PF network in control (WT) or Nkx2-5 +/ − (NK) hearts.

Article Snippet: Antibodies used in this study are specific to sheep GFP (1:500; AbD Serotec, Bio-Rad, Hercules, CA, USA), chick GFP (1:500; 2010, Aves), and goat Contactin-2 (1:100; AF1714 R&D Systems, Minneapolis, MN, USA), Donkey anti-goat-488 (1:500; A11055, Life Technologies), Donkey anti-goat-647 (1:250; A21447, Life technologies), Donkey anti-chick-488 (1:500; 703 545 155, Interchim), WGA-555 (1:500; Wheat germ agglutinin, Clinisciences).

Techniques: Expressing, Fluorescence, Immunostaining

Journal: Nature Communications

Article Title: Nkx2-5 defines distinct scaffold and recruitment phases during formation of the murine cardiac Purkinje fiber network

doi: 10.1038/s41467-020-19150-9

Figure Lengend Snippet: a and b Genetic tracing of Cx40+ cells labeled at E18.5 by Tam injection. Confocal images of whole-mount CNTN2-immunostaining of Cx40-CreERT2::R26R-YFP opened-left ventricle at P21. a ″ and b ″, Immunostaining for YFP and CNTN2 on control, or Nkx2-5 +/ − mice shows E18.5 Cx40 +-derived cells that participate to the PF network. Scale bars = 1 mm; a ’ and b ’ are high magnifications of left bundle branch; a ″ and b ″ are high magnifications of PF. c – e Strategy to induce conditional deletion of one copy of Nkx2-5 allele ( Nkx2-5 flox/+ ) using Cx40-CreERT2 line as inducible Cre driver. Deletion is performed at E18.5 when Cx40 + cells are mostly PF cells or E10.5 before the massive recruitment of late progenitors. R26R-YFP reporter line is used to compare the genetic tracing of Cx40 +-derived cells within P21 control, Nkx2-5 +/ − and Nkx2-5 flox/+ hearts ( N = 7 of each genotype for E18.5 and N = 3 for E10.5). e Confocal imaging of left PF network. Scale bars = 500 µm; arrowheads indicate non-conductive Cx40 +-derived cells.

Article Snippet: Antibodies used in this study are specific to sheep GFP (1:500; AbD Serotec, Bio-Rad, Hercules, CA, USA), chick GFP (1:500; 2010, Aves), and goat Contactin-2 (1:100; AF1714 R&D Systems, Minneapolis, MN, USA), Donkey anti-goat-488 (1:500; A11055, Life Technologies), Donkey anti-goat-647 (1:250; A21447, Life technologies), Donkey anti-chick-488 (1:500; 703 545 155, Interchim), WGA-555 (1:500; Wheat germ agglutinin, Clinisciences).

Techniques: Labeling, Injection, Immunostaining, Derivative Assay, Imaging