Journal: Science advances

Article Title: Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function.

doi: 10.1126/sciadv.adg0686

Figure Lengend Snippet: Fig. 3. Expression and cleavage of selected gelatinase substrates in TNF- α and unstimulated WT astrocyte cultures. (A) Immunofluorescence staining for MMP substrates, VCAM-1, NrCAM, agrin, NOTCH3, together with GFAP to mark astrocytes and DAPI; boxed areas are shown to the right at higher magnifications. Scale bars, 100 μm. (B) Silver-stained gels showing cleavage products of gelatinase substrates after overnight incubation without (0) or with 1:10 or 1:100 ratios of MMP-9:substrate or ADAM10:substrate. Arrows mark the positions of ADAM10 in samples. Asterisks mark specific cleavage products. Data are representative of two to three experiments.

Article Snippet: To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fcchimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl2, 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio).

Techniques: Expressing, Immunofluorescence, Staining, Incubation

Journal: Science advances

Article Title: Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function.

doi: 10.1126/sciadv.adg0686

Figure Lengend Snippet: Fig. 4. In vivo expression of MMP substrates, NrCAM and VCAM-1, in noninflamed (naïve) and EAE brains. WT brain sections were immunofluorescently stained for (A) GFAP to mark astrocytes, anti–laminin-γ1 chain antibody to mark BMs and perivascular cuffs, and NrCAM; DAPI marks all nuclei; scale bars, 100 μm; areas marked by the dotted lines are shown at higher magnifications in boxed areas. (B) Immunofluorescence staining for VCAM-1 and pan-laminin (Pan LM) in naïve and in early- and late- stage EAE brains; arrow marks VCAM-1 in CNS parenchyma at late-stage EAE; DAPI marks nuclei; scale bars, 100 μm (naïve) and 50 μm (early/late cuff). (C and D) Triple immunofluorescence staining for CD45, Pan LM, and VCAM-1 shows up-regulation of VCAM-1 around inflammatory cuffs, and its loss at this site where CD45 infiltration occurs (arrows), which correlates with sites of gelatinase activity as shown by in situ zymography (C, bottom) performed on consecutive sections; images to the far right in (C) show in situ hybridizations performed in the presence of the MMP inhibitor, 1,10-phenantrolin. Boxed area in (D) is shown at higher magnification in bottom panels; yellow asterisks mark vessel lumen. Scale bars, 100 μm (C) and 50 μm (D).

Article Snippet: To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fcchimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl2, 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio).

Techniques: In Vivo, Expressing, Staining, Immunofluorescence, Activity Assay, In Situ, Zymography

Journal: Science advances

Article Title: Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function.

doi: 10.1126/sciadv.adg0686

Figure Lengend Snippet: Fig. 6. NrCAM function on astrocytes. (A) Immunofluorescence staining of WT and DKO astrocyte-neuronal cocultures for vGlut to mark excitatory synapses and vGAT to mark inhibitory synapses, plus GFAP to mark astrocytes. Wavelet transformations of synapse stainings are shown in bottom panels; scale bars, 20 μm. (B) Corresponding statistical analysis of four experiments with separate culture preparations; data are expressed as relative frequency of GABAergic compared to glutamatergic synapses. Data are means ± SD with two replicates and seven to eight regions analyzed per experiment with each region comprising around 500 to 1000 synapses. Statistical analysis was Student’s t test; *P < 0.05. (C) Immunofluorescence staining of NrCAM, GFAP, and either MAP2 to mark neurons, vGlut, or vGAT in WT and DKO astrocyte- neuronal cocultures; scale bars, 10 μm.

Article Snippet: To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fcchimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl2, 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio).

Techniques: Immunofluorescence, Staining

Journal: Science advances

Article Title: Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function.

doi: 10.1126/sciadv.adg0686

Figure Lengend Snippet: Fig. 7. Identification of VCAM-1 and NrCAM in multiple sclerosis CSF samples and brain sections. (A) Representative gelatin gel zymography of CSF samples from patients with multiple sclerosis (MS1 to MS6) and age- and sex-matched somatoform controls (details in table S1). NGAL is neutrophil gelatinase–associated lipocalin. (B) ELISA for total MMP-9 in relapsing-remitting multiple sclerosis (RRMS) (n = 15) and somatoform (n = 15) CSF samples; statistical analyses were Mann-Whitney, **P < 0.005. The same CSF samples were tested in (C) ELISA for soluble VCAM-1 (sVCAM-1); data are expressed as change relative to somatoform controls. AU is arbitrary units. (D) Western blot for soluble NrCAM (sNrCAM); bar graph shows quantification of band intensities. Statistical analyses were Student’s t test (C) and Mann-Whitney (D); **P < 0.005 and ****P < 0.0001. (E) Double immunofluorescence staining for GFAP and VCAM-1 or NrCAM in normal-appearing white matter (NAWM), perivascular infiltrates in NAWM, and demyelinating lesions; scale bars, 100 μm. Arrows mark VCAM-1 expressed at the perivascular border and in the CNS parenchyma; bottom panels showing an astrocyte-expressing VCAM-1 in the demyelinating lesion at a higher magnification; scale bar, 25 μm.

Article Snippet: To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fcchimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl2, 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio).

Techniques: Zymography, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Western Blot, Double Immunofluorescence Staining, Expressing

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: Expression of CHL1 and NrCAM in pediatric neuroblastoma. Representative examples of CHL1 positive (A) and CHL-1 negative (B) (magnification x100) as well as NrCAM positive (C) and NrCAM negative (D) immunostaining (magnification x200).

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing, Immunostaining

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: Kaplan-Meier survival curves for overall and event-free survival. No association was found for CHL1-expression (A/B). Survival rates were better by trend in children with NrCAM positive tumors (C/D) but without statistical significance (p=0.07 and p=0.06).

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing

Journal: Journal of molecular neuroscience : MN

Article Title: Neural cell adhesion molecule NrCAM is expressed in the mammalian inner ear and modulates spiral ganglion neurite outgrowth in an in vitro alternate choice assay.

doi: 10.1007/s12031-014-0436-y

Figure Lengend Snippet: Fig. 1 Immunolabeling of NrCAM in the mouse cochlea at E14 and E17. Cell nuclei are in blue, NrCAM in red, Hes5 in green. a, c Overview of sagittal section of mouse head at the embryonic stage E14 and E17, stained with Hematoxylin/Eosine. Boxed is the cochlea. b The diagram of the embryonic OC. Source: http://syllabus.med.unc. edu. d, e Overview, NrCAM can be found in the KIO. lo (d, f) NrCAM can be detected in epithelial cells located in the embryonic cochlear duct at E14. e, g At E17 immunofluorescence images, NrCAM can be observed in the OC. Hes5 staining served as marker for embryonic cochlea. a, c Image by light microscopy, d–g images by immunofluorescence microscopy. Four mice per embryonic day were analyzed. Scale bar=50 μm

Article Snippet: NrCAM stripes were visualized using polyclonal rabbit antibodies against NrCAM (1:400; R&D Systems).

Techniques: Immunolabeling, Staining, Immunofluorescence, Marker, Light Microscopy, Microscopy

Journal: Journal of molecular neuroscience : MN

Article Title: Neural cell adhesion molecule NrCAM is expressed in the mammalian inner ear and modulates spiral ganglion neurite outgrowth in an in vitro alternate choice assay.

doi: 10.1007/s12031-014-0436-y

Figure Lengend Snippet: Fig. 2 The protein expression pattern of NrCAM in the mouse cochlea from P0 through P21. Cell nuclei are in blue, NrCAM in red, myosin 7A in green served as marker for HCs. a Diagram of the OC. Source: http://syllabus.med. unc.edu. b Negative control. c NrCAM labeling included the region of the IHC, OHC, and DC. d NrCAM labeling was slightly decreased. e Levels of NrCAM labeling is strong in the target zone of the IHC and OHC. f NrCAM labeling is strongest in the region of the OHC. g NrCAM labeling is strong at the IHC and OHC region. b–g Immunofluorescence microscopy images. Four mice per postnatal day were analyzed. Scale bar= 100 μm

Article Snippet: NrCAM stripes were visualized using polyclonal rabbit antibodies against NrCAM (1:400; R&D Systems).

Techniques: Expressing, Marker, Negative Control, Labeling, Immunofluorescence, Microscopy

Journal: Journal of molecular neuroscience : MN

Article Title: Neural cell adhesion molecule NrCAM is expressed in the mammalian inner ear and modulates spiral ganglion neurite outgrowth in an in vitro alternate choice assay.

doi: 10.1007/s12031-014-0436-y

Figure Lengend Snippet: Fig. 3 Quantitative analysis of termination by P5 rat SG neurites on alternating stripes of NrCAM (1, 5, or 10 μg/ml, respectively) versus PLL (5 μg/ml). Neurites showed a dose-dependent preference of NrCAM. Bars represent mean and SD of neurites/explants. *p<0.05, Mann–Whit- ney U test. 16–20 SG explants were analyzed per experimental condition

Article Snippet: NrCAM stripes were visualized using polyclonal rabbit antibodies against NrCAM (1:400; R&D Systems).

Techniques:

Journal: Journal of molecular neuroscience : MN

Article Title: Neural cell adhesion molecule NrCAM is expressed in the mammalian inner ear and modulates spiral ganglion neurite outgrowth in an in vitro alternate choice assay.

doi: 10.1007/s12031-014-0436-y

Figure Lengend Snippet: Fig. 4 Representative example of a P5 rat SG explant. Explants were cultured on an NrCAM (1, 5, or 10 μg/ml, respectively) versus PLL (5 μg/ml) alternating stripe pattern for 72 h and stained with an anti- neurofilament antibody (red) to visualize all SG neurites, and with an anti-NrCAM antibody (green) to visualize NrCAM. There was a tenden- cy for neurites to terminate upon and/or grow along the NrCAM substrate at NrCAM concentrations of 5 and 10 μg/ml. Stripe width 100 μm

Article Snippet: NrCAM stripes were visualized using polyclonal rabbit antibodies against NrCAM (1:400; R&D Systems).

Techniques: Cell Culture, Staining

Journal: Journal of molecular neuroscience : MN

Article Title: Neural cell adhesion molecule NrCAM is expressed in the mammalian inner ear and modulates spiral ganglion neurite outgrowth in an in vitro alternate choice assay.

doi: 10.1007/s12031-014-0436-y

Figure Lengend Snippet: Fig. 5 a Effects of NrCAM on the number of neurites extending from SG explants cultivated on increasing concentration of NrCAM. Number of neurites did not differ from control explants grown on PLL 5 μg/ml. Mean of neurites/explant±SD. b Effects of NrCAM on the average length of neurites per SG explant cultivated on increasing concentration of NrCAM. The average length of neurites did not differ from control explants grown on PLL 5 μg/ml. Mean length of neurites/ explant±SD. 16–20 SG explants were analyzed per experimental condition. c Representative example for P5 rat SG explants grown on uniform NrCAM or uniform PLL surface. Explants were cultured for 72 h and stained with an anti-neurofilament primary antibody to visualize all SG neurons

Article Snippet: NrCAM stripes were visualized using polyclonal rabbit antibodies against NrCAM (1:400; R&D Systems).

Techniques: Concentration Assay, Control, Cell Culture, Staining