mouse recombinant nrcam  (R&D Systems)

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