human neurocan recombinant protein  (R&D Systems)

Journal: bioRxiv

Article Title: Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.1101/2022.09.03.506497

Figure Lengend Snippet: NCAN is a major CSPG produced by developing cortical neurons. ( A ) Detection of the neoepitope of CSPGs after digestion with chondroitinase ABC (Chase). (B ) Immunoblotting of the neoepitope from undigested (Chase −) and digested (Chase +) cerebral cortex lysates prepared at E18. The arrowhead indicates the major CSPG at 130 kDa. ( C ) List of peptide fragments identified from the 130-kDa band. Positions indicate the amino acid number in full-length NCAN. ( D ) Domain structure of mouse NCAN. Ig: immunoglobulin-like domain, Link: hyaluronan-binding link module, EGF: epidermal growth factor-like repeat, CLD: C-type lectin domain, CS: chondroitin sulfate chain. ( E, F ) Developmental changes in NCAN expression from E13 to postnatal day (P) 42. The arrowheads indicate the full-length and N-terminal fragment of NCAN (E). Relative levels of the full-length (Full) and N-terminal fragment (N) of NCNA (F). Values are normalized to GAPDH and represented relative to P2. n = 3 for each point. Mean ± SD. ( G ) Expression levels of Ncan mRNA. Values are normalized to Gapdh and represented relative to E13. n = 3 for each point. Mean ± SD. ( H ) Expression of Ncan mRNA in GFP-labeled cells isolated on the indicated days after in utero electroporation. Values are normalized to Gapdh and represented relative to 0.6 days. n = 3 for each point. Mean ± SD. ( I ) Immunoblot analysis of NCAN in the cultured medium of primary cultured cortical neurons 1, 3, and 5 days after plating.

Article Snippet: Cover glasses were pre-coated with 20 μg/mL poly-L-ornithine (PLO) (Wako) at 37°C overnight and then overlaid with 0.1–10 μg/mL of recombinant human TNC (R&D), recombinant human NCAN (R&D), and HA (600–1200 kDa, PG research) at 37°C for 3 h. Embryonic brains were harvested 24 h after in utero electroporation with pCAG-GFP at E14 or E15 .

Techniques: Produced, Western Blot, Binding Assay, Expressing, Labeling, Isolation, In Utero, Electroporation, Cell Culture

Journal: bioRxiv

Article Title: Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.1101/2022.09.03.506497

Figure Lengend Snippet: Screening of the interacting partners of NCAN. ( A ) Schematic of the pull-down assay to identify NCAN-interacting partners. ( B ) List of the top 5 proteins identified by a LC-MS/MS analysis. ( C ) Domain structure of mouse TNC. EGF: epidermal growth factor-like repeat, FNIII: fibronectin type-III domain. ( D ) Detection of TNC in cerebral cortex lysates prepared from E13 to P42. ( E, F ) Expression of the TNC protein (E) and Tnc mRNA (F) during cortical development. Values are normalized to GAPDH and represented relative to E18 (E) or E13 (F). n = 3 for each point. Mean ± SD. ( G, H ) Localization of Tnc mRNA (G) and the TNC protein (H) in the E16 cerebral cortex. Scale bars represent 200 μm.

Article Snippet: Cover glasses were pre-coated with 20 μg/mL poly-L-ornithine (PLO) (Wako) at 37°C overnight and then overlaid with 0.1–10 μg/mL of recombinant human TNC (R&D), recombinant human NCAN (R&D), and HA (600–1200 kDa, PG research) at 37°C for 3 h. Embryonic brains were harvested 24 h after in utero electroporation with pCAG-GFP at E14 or E15 .

Techniques: Pull Down Assay, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: bioRxiv

Article Title: Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.1101/2022.09.03.506497

Figure Lengend Snippet: Radial glial cell-derived TNC interacts with the C-terminal half of NCAN. ( A ) Recombinant expression of the GFP-fused full-length, N-terminal half, and C-terminal half of NCAN. The culture medium of transfected HEK293 cells was digested with chondroitinase ABC (Chase) and analyzed by immunoblotting with an anti-GFP antibody. ( B ) Co-precipitation of TNC with the full-length and C-terminal half of NCAN. Interactions between NCAN and TNC disappeared following the addition of EDTA, but were not affected by Chase digestion. The N-terminal half of NCAN did not bind to TNC. ( C ) Alphafold2 prediction of mouse (m) NCAN-TNC complex. Five predicted complex models were generated using AlphaFold2 multimer implemented in ColabFold, and the best-predicted complex was shown. ( D , E ). Sequence alignments of CLD of mNCAN and rat (r) ACAN (D) and FNIII-3-5 of mTNC and rTNR (E). Circles under alignments indicate the key residues of the rACAN-TNR complex. Triangles over alignments indicate the residues in the interface of mNCAN-TNC. Green and magenta circles/triangles are the residues involved in the interaction between L4 loop of CLD and βC, F, G strands of FNIII-4 and β6, 7 strands of CLD and CC’ loop of FNIII-4, respectively. Black circles/triangles indicate residues involved in the interaction between CLD and FNIII-4-5 linker region/FG loop of FNIII-5.

Article Snippet: Cover glasses were pre-coated with 20 μg/mL poly-L-ornithine (PLO) (Wako) at 37°C overnight and then overlaid with 0.1–10 μg/mL of recombinant human TNC (R&D), recombinant human NCAN (R&D), and HA (600–1200 kDa, PG research) at 37°C for 3 h. Embryonic brains were harvested 24 h after in utero electroporation with pCAG-GFP at E14 or E15 .

Techniques: Derivative Assay, Recombinant, Expressing, Transfection, Western Blot, Generated, Sequencing

Journal: bioRxiv

Article Title: Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.1101/2022.09.03.506497

Figure Lengend Snippet: Alphafold2 prediction of NCAN-TNC complex. ( A ) Model confidence of NCAN-TNC complex. Each residue is colored by pLDDT score, and high pLDDT score indicates high accuracy. ( B ) Crystal structure of rat (r) ACAN-TNR complex (PDB ID: 1TDQ). Each domain is colored the same as in . ( C ) Sequence alignments of fourteen FNIII of mTNC. Green letters indicate residues involved in the interaction between FNIII-4 and CLD of NCAN.

Article Snippet: Cover glasses were pre-coated with 20 μg/mL poly-L-ornithine (PLO) (Wako) at 37°C overnight and then overlaid with 0.1–10 μg/mL of recombinant human TNC (R&D), recombinant human NCAN (R&D), and HA (600–1200 kDa, PG research) at 37°C for 3 h. Embryonic brains were harvested 24 h after in utero electroporation with pCAG-GFP at E14 or E15 .

Techniques: Sequencing

Journal: bioRxiv

Article Title: Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.1101/2022.09.03.506497

Figure Lengend Snippet: HA, NCAN, and TNC from the ternary complex in the developing cerebral cortex. ( A ) Triple staining of the E17 mouse cerebral cortex with HA (cyan), NCAN (magenta), and TNC (green). Right panels are high magnification images showing the co-localization of the three components in the upper part of the SP/IZ, but not in the CP or VZ. ( B ) Localization of HA, NCAN, and TNC in the cerebral cortex two days after an intraventricular injection of PBS or hyaluronidase (HAase) at E14. The normalized fluorescence intensity profiles of HA, NCAN, and TNC are shown on the right of each image. N = 5 mice for each group. Mean ± SD (shaded area). ( C ) Immunoblot analysis of NCAN and TNC in cerebral cortex lysates two days after an intraventricular injection of PBS or HAase. Protein levels were represented relative to the PBS-injected group. N = 4 mice per group. Mean ± SD; *p < 0.01; the Student’s t -test. ( D ) Distribution patterns of HA in the E16 cerebral cortex of WT and DKO mice. The fluorescence intensity profile is shown on the right. N = 11 mice for each group. Mean ± SD (shaded area). ( E ) Model for forming the ternary complex of NCAN, HA, and TNC. Scale bars represent 100 μm (left panels in A), 5 μm (right panels in A), and 50 μm (B, D).

Article Snippet: Cover glasses were pre-coated with 20 μg/mL poly-L-ornithine (PLO) (Wako) at 37°C overnight and then overlaid with 0.1–10 μg/mL of recombinant human TNC (R&D), recombinant human NCAN (R&D), and HA (600–1200 kDa, PG research) at 37°C for 3 h. Embryonic brains were harvested 24 h after in utero electroporation with pCAG-GFP at E14 or E15 .

Techniques: Staining, Injection, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.1101/2022.09.03.506497

Figure Lengend Snippet: Characterization of double knockout (DKO) mice for NCAN and TNC. ( A ) Immunoblot analysis of TNC and NCAN from WT and DKO mouse cerebral cortex lysates digested with or without chondroitinase ABC (Chase). ( B ) Immunostaining of NCAN and TNC on coronal sections of WT and DKO mouse brains at E17. Scale bars represent 200 μm. ( C) No significant difference in cerebrum weights between WT and DKO mice at E16. N = 5 mice per group. Mean ± SD; ns > 0.05; the Student’s t -test. ( D ) No significant difference in the amount of HA in the cerebrum between WT and DKO mice at E16. N = 5 mice per group. Mean ± SD; ns > 0.05; the Student’s t -test.

Article Snippet: Cover glasses were pre-coated with 20 μg/mL poly-L-ornithine (PLO) (Wako) at 37°C overnight and then overlaid with 0.1–10 μg/mL of recombinant human TNC (R&D), recombinant human NCAN (R&D), and HA (600–1200 kDa, PG research) at 37°C for 3 h. Embryonic brains were harvested 24 h after in utero electroporation with pCAG-GFP at E14 or E15 .

Techniques: Double Knockout, Western Blot, Immunostaining

Journal: bioRxiv

Article Title: Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.1101/2022.09.03.506497

Figure Lengend Snippet: Disruption of the ternary complex retards neuronal migration. ( A ) Experimental model for in utero cell labeling. ( B, C ) Localization of TNC (B, magenta), NCAN (B, C, cyan), and HA (C, magenta) around GFP-labeled bipolar neurons (green) in the upper SP/IZ at E17, three days after in utero labeling. Orthogonal views taken along the white dashed lines showed the contact between the ternary complex and the surface of bipolar neurons, as indicated by the arrows. ( D ) Low magnification images of FT-labeled cells (green) in WT and DKO mouse coronal sections at E16. Nuclei were counterstained with DAPI (blue). M: medial; L: lateral. ( E, F ) Radial distribution of FT-labeled cells (green) in the lateral (E) and medial (F) cortex of WT and DKO mice at E16. A quantitative analysis of migration profiles across the cortex is shown on the right. The cerebral cortex is divided into five equal areas (bins 1–5) from the pia to the ventricle, and the proportion of FT-labeled cells in each bin was calculated. N = 19–20 mice per group. Mean ± SD; *p < 0.05; the Student’s t -test. ( G, H ) Distribution of FT-labeled cells (green) in the lateral (G) and medial (H) cortices two days after an injection of PBS or hyaluronidase (HAase) at E14. Migration profiles were analyzed as in (E, F). N = 9–10 mice per group. Mean ± SD; *p < 0.05; the Student’s t -test. Scale bars represent 2 μm (B), 200 μm (D), and 50 μm (E-H).

Article Snippet: Cover glasses were pre-coated with 20 μg/mL poly-L-ornithine (PLO) (Wako) at 37°C overnight and then overlaid with 0.1–10 μg/mL of recombinant human TNC (R&D), recombinant human NCAN (R&D), and HA (600–1200 kDa, PG research) at 37°C for 3 h. Embryonic brains were harvested 24 h after in utero electroporation with pCAG-GFP at E14 or E15 .

Techniques: Migration, In Utero, Labeling, Injection

Journal: bioRxiv

Article Title: Assembly of neuron- and radial glial cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.1101/2022.09.03.506497

Figure Lengend Snippet: TNC promotes the neurite outgrowth and morphological maturation of cortical neurons. ( A ) Experimental model for in utero cell labeling and the primary culture. ( B ) Morphological stages of primary cultured cortical neurons. ( C ) Representative images of GFP-labeled neurons cultured for 1 to 3 days on cover glasses coated with the control substrate, poly-L-ornithine (POL), and 10 μg/mL of TNC, HA, and NCAN. ( D ) The percentage of neurons with each morphological stage after culturing on the indicated substrate for 2 and 3 days. ( E-G ) The total length of neurites (E), length of the longest neurite (F), and the number of neurites (G) of neurons cultured on the indicated substrate for 2 and 3 days. N = 28–46 cells per condition. Mean ± SD; *p < 0.05 vs POL; Dunnett’s test. Scale bars represent 20 μm.

Article Snippet: Cover glasses were pre-coated with 20 μg/mL poly-L-ornithine (PLO) (Wako) at 37°C overnight and then overlaid with 0.1–10 μg/mL of recombinant human TNC (R&D), recombinant human NCAN (R&D), and HA (600–1200 kDa, PG research) at 37°C for 3 h. Embryonic brains were harvested 24 h after in utero electroporation with pCAG-GFP at E14 or E15 .

Techniques: In Utero, Labeling, Cell Culture

Journal: Experimental neurology

Article Title: Increased matrix metalloproteinase levels and perineuronal net proteolysis in the HIV-infected brain; relevance to altered neuronal population dynamics

doi: 10.1016/j.expneurol.2019.113077

Figure Lengend Snippet: MMPs cleave the perineuronal net proteins aggrecan and brevican. Active recombinant matrix metalloproteinases were incubated with recombinant aggrecan and brevican. In vitro digests revealed that A. aggrecan (Acan) and B. brevican (Bcan) are both cleaved by MMP-3 and MMP-13, as demonstrated by the appearance of the indicated cleavage fragments. Enzymatic cleavage is prevented by addition of the broad-spectrum MMP inhibitor GM6001.

Article Snippet: Recombinant human aggrecan (R&D, catalog # 1220-PG-025) was used at a concentration of 62.5 μg/mL and recombinant human brevican (R&D, catalog # 5800-NC-050) was used at a concentration of 50 μg/mL.

Techniques: Recombinant, Incubation, In Vitro