Journal: Autophagy

Article Title: The autophagy-inducing kinases, ULK1 and ULK2, regulate axon guidance in the developing mouse forebrain via a noncanonical pathway

doi: 10.1080/15548627.2017.1386820

Figure Lengend Snippet: Abnormal axonal fasciculation in the Ulk1/2-deficient animals is associated with mislocalization of CNTN2 in the projection neurons. (A) Neuronal cell adhesion molecule distribution was unaltered in the ulk1/2-DKO brains compared to that in the controls. (B) The intensity of CNTN2 immunostaining (red) was dramatically decreased in distal CTAs of the ulk1/2-DKO brain at E18.5. All of the sections were counterstained with CHL1 cell adhesion molecule (green). (C) Western blot analyses of the extracts prepared from the cortex (proximal CTAs) and striatum (distal CTAs) confirmed normal expression of neuronal cell adhesion molecule but significantly decreased CNTN2 levels in the striatum. (D and E) Quantification of the NCAM1 and CNTN2 levels in control and ulk1/2-DKO cortex and striatum. Abbreviations: Cnt, controls; NCAM1, neuronal cell adhesion molecule 1; ns, not significant.*P < 0.05. Scale bars: 200 µm.

Article Snippet: After incubation with a 5% skim-milk block, blots were probed with antibodies directed against the following targets: ULK1 (Sigma Aldrich, A7481), SQSTM1 (Sigma Aldrich, P0067), LC3B (Novus Biologicals, NB100–2220), ATG13 (Sigma Aldrich, SAB4200100), RB1CC1 (Cell Signaling Technology, 12436), ATG14 (MBL, PD026), CNTN2 (R&D, AF4439), NCAM1 (Developmental Studies Hybridoma Bank, 5B8), and GAPDH (Sigma Aldrich, G9545).

Techniques: Immunostaining, 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 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: The Journal of Clinical Investigation

Article Title: In vivo visualization and molecular targeting of the cardiac conduction system

doi: 10.1172/JCI156955

Figure Lengend Snippet: ( A ) Antibody-dye conjugate (mCntn2-800) consists of a near-infrared (NIR) dye conjugated to an antibody against the CCS-specific surface marker Cntn2 (contactin 2). ( B ) Experimental work flow. ( C ) Whole-body biodistribution of other tissue types, showing expected clearance within the liver, bladder, and kidneys and notable absence from the brain. ( D ) Whole mouse heart ( n = 3) from a wild-type (WT) mouse injected 3 days prior with mCntn2-800, in posterior-anterior (PA) and right lateral (RL) views. Atria are outlined in white and cardiac chambers are listed. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Top: Brightfield. Bottom: NIR signal demonstrating labeling of the CCS (blue→red = lowest→highest signal). Mean signal to background ratio (SBR) is indicated. ( E ) Measured intervals (in ms) from sedated surface electrocardiograms (ECGs) including PR, QRS, QTc, and RR in WT mice prior to (day 0 = baseline, n = 12) and daily (day 1 n = 3, day 2 n = 9; after injection) following a single tail vein injection of mCntn2-800. Intervals (mean ± SD) on a given day after injection were compared to each mouse’s preinjection control baseline (day 0) using 1-way ANOVA with Tukey’s post hoc test. ( F – H ) Heart sections from a WT mouse injected 2 days prior with mCntn2-800. CCS components labeled with mCntn2-800 (purple) and costained with antibodies targeting known markers of the CCS, including anti-Hcn4 (red, SAN and His) or anti-Cx40 (green, PF). DAPI (blue, nuclei). LBB/RBB, left and right bundle branches; His, His bundle; PF, Purkinje fibers; SAN, sinoatrial node; VM, ventricular myocardium. Scale bars: 10 mm ( C ), 5 mm ( D ), and 100 μm ( F – H ).

Article Snippet: Described antibody-dye conjugates consist of commercially acquired anti-Cntn2 goat polyclonal antibody (AF4439) and anti-NPTN goat polyclonal antibody (AF5360) (both R&D Systems) that have been covalently conjugated to a benign, NIR dye (IRDye800CW NHS ester; LI-COR Biosciences, 929-70020) using company specifications.

Techniques: Marker, Injection, Labeling

Journal: The Journal of Clinical Investigation

Article Title: In vivo visualization and molecular targeting of the cardiac conduction system

doi: 10.1172/JCI156955

Figure Lengend Snippet: ( A ) Experimental workflow: wild-type mice ( n = 3) received a tail vein injection of mCntn2-800 and after 48 hours were sedated and received a sternotomy and cardiac incisions including a right atriotomy and right ventriculotomy to simulate a surgical scenario. Live imaging of the heart with a FLARE Intraoperative Near-Infrared (NIR) Fluorescence Imaging System. ( B and C ) Heart with visible sinoatrial node (SAN), atrioventricular node/His bundle (AVN/His), and Purkinje fiber (PF) network. Left: Color image of ex vivo heart. Right: mCntn2-800 NIR signal (green). Middle: Merged image of color image and NIR (green) signal. ( D ) Heart sections from the same heart, demonstrating mCntn2-800 signal (cyan) labeling the SAN, as costained with anti-Cntn2 (red) immunostaining. ( E ) Magnified region in SAN indicated by the white box in D . Right atrial (RA) tissue labeled with anti-Cx43 (purple). DAPI (blue, nuclei). IVS, interventricular septum; RV, right ventricle; SAN, sinoatrial node; SVC, superior vena cava. Scale bars: 5 mm ( B and C ), 300 μm ( D ), and 100 μm ( E ).

Article Snippet: Described antibody-dye conjugates consist of commercially acquired anti-Cntn2 goat polyclonal antibody (AF4439) and anti-NPTN goat polyclonal antibody (AF5360) (both R&D Systems) that have been covalently conjugated to a benign, NIR dye (IRDye800CW NHS ester; LI-COR Biosciences, 929-70020) using company specifications.

Techniques: Injection, Imaging, Fluorescence, Ex Vivo, Labeling, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: In vivo visualization and molecular targeting of the cardiac conduction system

doi: 10.1172/JCI156955

Figure Lengend Snippet: ( A ) Human anti-CNTN2 Fab antibody was biotinylated and conjugated to streptavidin-linked saporin, a cell toxin (hCNTN2-Sap). ( B ) Wild-type mice received a single tail vein injection of either hCNTN2-Sap (100 μg) ( n = 6) or Control-Sap (100 μg nonspecific human IgG similarly conjugated to saporin) ( n = 6). Mice received electrocardiograms (ECGs) on day 0 (baseline) and daily following injection with control or hCNTN2-Sap. On day 2, hearts were harvested, fixed, and immunostained. ( C ) Representative ECG tracings ( n = 6 per condition). ( D ) By day 2, mice injected with hCNTN2-Sap demonstrated marked conduction abnormalities, including prolonged PR, QRS, and RR intervals as compared with mice injected with Control-Sap (mean intervals in ms ± SD). Gray bar, QTc interval corrected for QRS intervals. Statistical analyses using 2-way ANOVA with Tukey’s post hoc test. ( E ) Consistent with targeted cell death, immunofluorescence of the CCS (red) showed subtotal loss of CCS cells as shown within the His bundle (His), right and left bundle branches (RBB/LBB) as stained by anti-Cntn2. His, His bundle; IVS, interventricular septum; LBB, left bundle branch; RBB, right bundle branch. Scale bar: 50 μm.

Article Snippet: Described antibody-dye conjugates consist of commercially acquired anti-Cntn2 goat polyclonal antibody (AF4439) and anti-NPTN goat polyclonal antibody (AF5360) (both R&D Systems) that have been covalently conjugated to a benign, NIR dye (IRDye800CW NHS ester; LI-COR Biosciences, 929-70020) using company specifications.

Techniques: Injection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Nhlh1 and Nhlh2, a global transcriptional mechanism regulating commissural axon projection via activating Robo3

doi: 10.1101/2022.09.23.509112

Figure Lengend Snippet: Lack of ventral commissures in the spinal cord, hindbrain and midbrain in Nhlh1 and Nhlh2 double mutant. Coronal sections from the spinal cord (A), caudal hindbrain (B), rostral hindbrain (C), and midbrain (D) at E11.5 were subjected to Tag1 and NF double IHC. The Tag1 signals label the commissural axons and the NF signals show the general axonal patterns. The DAPI counterstaining indicate the overall cytoarchitecture. Comparisons were made between the control genotype ( Nhlh1 +/+ Nhlh2 +/m ) (n = 3) and the double mutant ( Nhlh1 m/m Nhlh2 m/m ) (n = 3). The Tag1 and NF merged images in the bottom panel are high magnification images of the ventral commissure regions. Filled arrows show the ventral commissures and the hollow arrows show the lack of ventral commissures. Scale bars: 200 μm in low magnification images in (A), (B), (C) & (D); 200 μm in high magnification images in (A), (B), (C) & (D).

Article Snippet: The primary antibodies used were: rabbit anti-Barhl1 polyclonal antibody (Atlas Antibodies, HPA004809, Sigma, 1:500), goat anti-Robo3 polyclonal antibody (R&D Systems, AF3076, 1:200), goat anti-Tag1 polyclonal antibody (R&D Systems, AF4439, 1:500), mouse anti-Neurofilament-160KD monoclonal antibody (clone: RMO-270, Zymed, 13-0700, 1:500), rat anti-L1CAM monoclonal antibody (clone 324, Chemicon, MAB5272, 1:400), goat anti-DCC polyclonal antibody (Santa Cruz Biotechnology, sc-6535, 1:200), rabbit anti-FoxP2 polyclonal antibody (abcam, ab16046, 1:1000), mouse anti-Brn3a monoclonal antibody (clone: 5A3.2, Millipore, MAB1585, 1;200), chick anti-GFP polyclonal antibody (abcam, ab13970, 1:1500).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Nhlh1 and Nhlh2, a global transcriptional mechanism regulating commissural axon projection via activating Robo3

doi: 10.1101/2022.09.23.509112

Figure Lengend Snippet: Commissure formation does not appear to be affected in Nhlh1 and Nhlh2 single mutant. Commissural axons were labelled by Tag1 IHC, and the overall axonal patterns were labelled by NF IHC. Ventral commissure appears comparable between the control genotype of Nhlh1 +/+ Nhlh2 +/m (n = 2), and the mutant genotypes of Nhlh1 +/m Nhlh2 m/m (n = 2) and Nhlh1 m/m Nhlh2 +/m (n = 2), in the spinal cord (A), caudal hindbrain (B), rostral hindbrain (C), and midbrain (D). Higher magnification images of the commissural region across the ventral midline are shown in the bottom panels. Scale bars: 100μm in (A); 200 μm in (B), (C) & (D).

Article Snippet: The primary antibodies used were: rabbit anti-Barhl1 polyclonal antibody (Atlas Antibodies, HPA004809, Sigma, 1:500), goat anti-Robo3 polyclonal antibody (R&D Systems, AF3076, 1:200), goat anti-Tag1 polyclonal antibody (R&D Systems, AF4439, 1:500), mouse anti-Neurofilament-160KD monoclonal antibody (clone: RMO-270, Zymed, 13-0700, 1:500), rat anti-L1CAM monoclonal antibody (clone 324, Chemicon, MAB5272, 1:400), goat anti-DCC polyclonal antibody (Santa Cruz Biotechnology, sc-6535, 1:200), rabbit anti-FoxP2 polyclonal antibody (abcam, ab16046, 1:1000), mouse anti-Brn3a monoclonal antibody (clone: 5A3.2, Millipore, MAB1585, 1;200), chick anti-GFP polyclonal antibody (abcam, ab13970, 1:1500).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Nhlh1 and Nhlh2, a global transcriptional mechanism regulating commissural axon projection via activating Robo3

doi: 10.1101/2022.09.23.509112

Figure Lengend Snippet: Commissure-less phenotype in the Nhlh1 and Nhlh2 double mutant persisted into later developmental stages. Commissure formation was analyzed either by Tag1 or NF IHC. Lack of commissure formation continued to be observed in E13.5 spinal cord (A) (n = 2 for each genotype), E16.5 spinal cord (B) (n = 2 for each genotype), E13.5 hindbrain (C) (n = 2 for each genotype) and E13.5 midbrain (D) (n = 2 for each genotype). Scale bars: 200 μm in (A); 100 μm in (B); 400 μm in (C) & (D).

Article Snippet: The primary antibodies used were: rabbit anti-Barhl1 polyclonal antibody (Atlas Antibodies, HPA004809, Sigma, 1:500), goat anti-Robo3 polyclonal antibody (R&D Systems, AF3076, 1:200), goat anti-Tag1 polyclonal antibody (R&D Systems, AF4439, 1:500), mouse anti-Neurofilament-160KD monoclonal antibody (clone: RMO-270, Zymed, 13-0700, 1:500), rat anti-L1CAM monoclonal antibody (clone 324, Chemicon, MAB5272, 1:400), goat anti-DCC polyclonal antibody (Santa Cruz Biotechnology, sc-6535, 1:200), rabbit anti-FoxP2 polyclonal antibody (abcam, ab16046, 1:1000), mouse anti-Brn3a monoclonal antibody (clone: 5A3.2, Millipore, MAB1585, 1;200), chick anti-GFP polyclonal antibody (abcam, ab13970, 1:1500).

Techniques: Mutagenesis

Journal: Journal of molecular and cellular cardiology

Article Title: Partial and complete loss of myosin binding protein H-Like cause cardiac conduction defects

doi: 10.1016/j.yjmcc.2022.04.012

Figure Lengend Snippet: (A) Immunofluorescence microscopy of 5 μm thick frozen sections of wild type mouse hearts stained with MyBP-HL (red) and the ventricular conduction system marker contactin-2 (Cntn2). Panels show: low (i) and high (ii) magnification images of the interventricular septum at the base of the heart where atrial tissue enters the ventricle and MyBP-HL staining overlaps with contactin-2 in the ventricle. (iii) A contactin-2 stained Purkinje fiber in the RV endocardial wall shows co-staining with MyBP-HL in some ventricular conduction system cardiomyocytes. (iv) MyBP-HL-stained ventricular cardiomyocytes on the RV epicardium with no associated contactin-2 staining. (B) Immunofluorescence microscopy of 5 μm thick frozen sections of Mybphl heterozygous mouse hearts stained with MyBP-HL (red) and the ventricular conduction system marker contactin-2 (green). Panels show: low (i) and high (ii) magnification images of the interventricular septum at the base of the heart where atrial tissue enters the ventricle and MyBP-HL staining overlaps with contactin-2 in the ventricle. (iii) A large cluster of MyBP-HL positive cells in the LV septum with no associated contactin-2 staining. (iv) A multicellular cluster of MyBP-HL positive cells in the septum with overlapping contactin-2 staining. Female hearts.

Article Snippet: Primary antibodies were used against MyBP-HL (Pocono, custom), epitope and antibody validation in , cMyBP-C (Santa Cruz E7), contactin-2 (R&D Systems AF4439) [ 21 ], ryanodine receptor 2 (AbCam GR3250452–2).

Techniques: Immunofluorescence, Microscopy, Staining, Marker

Journal: Journal of molecular and cellular cardiology

Article Title: Partial and complete loss of myosin binding protein H-Like cause cardiac conduction defects

doi: 10.1016/j.yjmcc.2022.04.012

Figure Lengend Snippet: (A) Right ventricular free wall region from a WT heart stained with MyBP-HL (red), with expanded view of the RV free wall showing individual MyBP-HL-positive foci (arrows). (B) Right ventricular free wall region from a Mybphl heterozygous heart stained with MyBP-HL and contactin-2 show fewer and larger MyBP-HL foci. Speckled red background signal in panels A and B are off target staining from this antibody that occurs perinuclearly throughout the heart. (C) Quantification of the numbers of MyBP-HL foci per section. (D) Quantification of the percentage of MyBP-HL foci that were found in the RV free wall. N = average foci count per slide from 4 – 6 sections/slide. N = 7 WT, 7 Het slides. Female hearts. * = P< 0.05 by two-tailed t-test

Article Snippet: Primary antibodies were used against MyBP-HL (Pocono, custom), epitope and antibody validation in , cMyBP-C (Santa Cruz E7), contactin-2 (R&D Systems AF4439) [ 21 ], ryanodine receptor 2 (AbCam GR3250452–2).

Techniques: Staining, Two Tailed Test

Journal: Journal of molecular and cellular cardiology

Article Title: Partial and complete loss of myosin binding protein H-Like cause cardiac conduction defects

doi: 10.1016/j.yjmcc.2022.04.012

Figure Lengend Snippet: (A) Reconstructed images from 5 μm step size Z-stacks taken using lightsheet microscopy of a WT perinatal day 5 mouse heart immunostained for MyBP-HL (red) and contactin-2 (Cntn2) (green). Yellow scale volume 500 μm/side. Rendering of surface feature details (right panels) of the MyBP-HL stained atria and contactin-2 stained ventricular conduction system, as well as single spot renderings of MyBP-HL positive ventricular foci. (B) A histogram of the percentage of the shortest distance from each MyBP-HL spot to the contactin-2 surface in Mybphl WT, Het, and Null hearts showed enrichment of MyBP-HL spots near the contactin-2 surface in WT mice. (C) The mean shortest distance between MyBP-HL spots and the contactin-2 surface is significantly increased in Het and Null hearts. (D) WT hearts show most MyBP-HL spots are located within 100 μm of the contactin-2 surface. N = 3 WT, 3 Het, 4 Null. Mixed sex litters. * = p > 0.05 by One-Way ANOVA.

Article Snippet: Primary antibodies were used against MyBP-HL (Pocono, custom), epitope and antibody validation in , cMyBP-C (Santa Cruz E7), contactin-2 (R&D Systems AF4439) [ 21 ], ryanodine receptor 2 (AbCam GR3250452–2).

Techniques: Microscopy, Staining