Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Axonal Regrowth after Spinal Cord Injury via Chondroitinase and the Tissue Plasminogen Activator (tPA)/Plasmin System

doi: 10.1523/JNEUROSCI.3339-11.2011

Figure Lengend Snippet: a) Representative immunoblots of NG2 and neurocan protein expressions in WT and tPA KO sham and 14 day SCI homogenates incubated with ChABC for 3 hrs at 37°C and probed with rabbit anti-NG2 and mouse anti-Neurocan antibodies. Protein expression could not be evaluated after Pen treatment in vitro using western blotting since the presence of GAG chains resulted in a non-distinct protein band, not shown. α-tubulin was used as a loading control. b, c) Protein bands were quantified by densitometry in ImageJ and data normalized against α-tubulin loading control and total protein levels. One Way ANOVA was used to compare all groups. Significant ANOVA was followed by post hoc Holm Sidak test. # indicates significant post hoc differences at p<0.001 (n=3). (d-f) 14 days sham or contusion injured WT and tPA KO mice received Pen or ChABC injection (50U/ml × 1μl). Two days later spinal cords were isolated, perfused with PFA and 18μm sagittal sections prepared. Spinal cord sections were then triple-stained for GFAP (red) and 4’-6-Diamidino-2-phenylindole (DAPI, blue, nuclear marker) and various CSPG protein antigens in green b) NG2 (Chemicon), d) phosphacan (3F8; DSHB) f) neurocan (1F6; DSHB), and images captured with a Zeiss confocal microscope using LSM 510 Meta Software. Dashed line indicates border of injury region. Images are representative of 3-4 biological replicates per group. Scale bar=100μm. g) ImageJ software (NIH) was used to calculate mean intensity/area for each group around the injury border (identified by GFAP dead and reactive regions). t-test was used to compare within each treatment and one Way ANOVA to compare within genotype (n=3-4). Significant ANOVA values were followed by post hoc Holm Sidak. Brackets with asterisks indicate significance due to t-test or post hoc analyses with a minimum of p<0.05. h) Western blot of NG2 protein (gift from Dr. Joel Levine) incubated with ChABC and treated with recombinant plasminogen and different concentrations of uPA or tPA for 3 hours at 37°C, followed by incubation with rabbit anti-NG2 antibody. ChABC cleaved NG2 protein can be seen at 250kd and 148kd (arrowheads). i) Western blot of pure CSPG protein (CC117; Chemicon) incubated without (top blot) or with ChABC (bottom blot), pure plasminogen, uPA or tPA at 500U/ug and probed for neurocan protein (1F6, DSHB). Plasminogen protein levels (anti-mouse plasminogen1/1000, Millipore) were also assessed (middle blot). j) Western blot of sham or SCI protein extracts from WT and tPA KO animals treated with Pen or ChABC. The blots were probed with GFAP (Abcam), Iba1 (not shown) and α-tubulin.

Article Snippet: Sections were sequentially blocked with mouse blocking serum (Vector lab) and goat serum, and then briefly incubated in mouse diluent before probing with one of the following primary antibodies for 30 minutes at room temperature: CSPG (1/200, CS56; Sigma), NG2 (1/1000, rabbit anti-NG2), Neurocan (1/50, anti-mouse 1F6; DSHB), Phosphacan (1/100, mouse-3F8; DSHB), or Chondroitin-4-sulfate (C4S, mouse-MAB2030, 1:1000; Chemicon).

Techniques: Western Blot, Incubation, Expressing, In Vitro, Control, Injection, Isolation, Staining, Marker, Microscopy, Software, Recombinant

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Axonal Regrowth after Spinal Cord Injury via Chondroitinase and the Tissue Plasminogen Activator (tPA)/Plasmin System

doi: 10.1523/JNEUROSCI.3339-11.2011

Figure Lengend Snippet: tPA activity was visualized in media and cell lysates from embryonic day 15 WT and tPA KO cortical neurons using zymography (a), and measured using quantitative amidolytic assay (b). Pure recombinant tPA was used as positive control and can be seen at 68kd in (a). Significantly higher levels of tPA activity were measured in WT cortical neuron cell lysates compared to all other groups (n=3). One Way ANOVA was used to compare all groups. Significant ANOVA was followed by post hoc Holm Sidak test. # indicates significant post hoc differences at p<0.001. (c). Western Blot of 14 days SCI extracts from WT and tPA KO mice incubated with protease inhibitor cocktail (PIC) (1x; Sigma), and/or ChABC (0.5U/ml) for 3 hours at 37°C and probed for Neurocan protein and α-tubulin. (d) Spinal cord homogenates from sham or 14 day contusion injured mice were treated with Pen or ChABC (0.5U/ml) in vitro for 3 hours at 37°C. Embryonic day 15 WT and tPA KO cortical neurons were then plated on spinal cord homogenates of the same genotype. 2 days later, cultures were fixed with PFA and stained for intact CSPG (red; CS56; Sigma), and β-tubulin III (green; Tuj1; Covance) Scale bar = 100μm. Higher magnification images of a neuron in each group (indicated by an arrow in β-tubulin III staining) are provided in the lower left box of each image panel. Scale bar= 20μm. (e) Embryonic day 15 tPA KO cortical neurons were grown on 14 day contusion-injured tPA KO SCI homogenates previously co-treated with Pen and plasmin or ChABC and plasmin for 3 hours at 37°C. 2 days later, cultures were fixed with PFA and stained for intact CSPG (red), and β-tubulin III (green). Scale bar = 100μm. Higher magnification image of a neuron in each group (indicated by an arrow in β-tubulin III staining) is provided in the lower left box of each image panel. tPA KO cortical neurons grown on tPA KO SCI homogenates co-treated with ChABC and plasmin in vitro showed significantly higher neurite outgrowth compared with neurons grown on tPA KO SCI homogenates co-treated with Pen and plasmin. Scale bar= 20μm. Images are representative of 3 biological replicates per group. (f) Neurite Tracer plug-in in ImageJ was used to quantify 400-600 neurons per biological replicate. Data were calculated as neurite length/neuron, normalized and plotted as a percentage of the cortical neuron only group of the same genotype (n=3). One Way ANOVA was used for comparisons. Significant ANOVA was followed by post hoc Holm Sidak test. Brackets with asterisk indicate significant post hoc differences. Compared to neurite outgrowth on SCI homogenates from WT and tPA KO mice treated with Pen in vitro, p=0.031 and p=0.039 for neurite outgrowth on the same SCI homogenate samples from WT and tPA KO mice treated with ChABC in vitro. When compared within ChABC treated group, cortical neurons on WT SCI homogenates grew more processes than cortical neurons on tPA KO SCI homogenates (p<0.001). Furthermore, tPA KO cortical neurons grown on tPA KO SCI homogenates treated with ChABC and plasmin (0.06U/ml) in vitro show significantly higher neurite outgrowth when compared with neurons on tPA KO SCI homogenates treated with ChABC (p<0.001) or Pen Plasmin in vitro (p=0.002).

Article Snippet: Sections were sequentially blocked with mouse blocking serum (Vector lab) and goat serum, and then briefly incubated in mouse diluent before probing with one of the following primary antibodies for 30 minutes at room temperature: CSPG (1/200, CS56; Sigma), NG2 (1/1000, rabbit anti-NG2), Neurocan (1/50, anti-mouse 1F6; DSHB), Phosphacan (1/100, mouse-3F8; DSHB), or Chondroitin-4-sulfate (C4S, mouse-MAB2030, 1:1000; Chemicon).

Techniques: Activity Assay, Zymography, Recombinant, Positive Control, Western Blot, Incubation, Protease Inhibitor, In Vitro, Staining

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: GAG-modified neurocan blocks chABC-induced decrease of perisomatic synaptic puncta in organotypic brain slices. ( A ) Immunostaining of neuronal soma (NeuN), a parvalbumin-positive interneuron (tdTomato), and a perineuronal net (WFA) in DIV14 organotypic brain slice culture. Scale bar = 10 μm. ( B ) WFA labeling of perineuronal nets in control penicillinase and chABC-treated brain slices. Scale bar = 30 μm. ( C ) Representative image of perisomatic synapses (tdTomato) in control penicillinase or chABC-treated slice cultures. Representative perisomatic puncta around a single soma are indicated with arrowheads. Scale bar = 10 μm. ( D ) Quantification of the mean number of perisomatic synaptic puncta per soma (n = 30 soma/condition, 3 animals per condition, t-test, *p < 0.05). ( E ) NCAM was immunoprecipitated from brain lysates, followed by immunoblotting with antibodies against neurocan, versican, or aggrecan/brevican (using an antibody raised against shared epitope). ( F ) Slices were treated with control penicillinase or chABC as in ( C ) followed by rescue with neurocan or tenascin-R. Quantification of the mean number of perisomatic synapses per soma was performed (>90 soma per mouse per condition, n = 3 mice, two-way ANOVA with Bonferonni post-hoc testing, *p < 0.05). ( G ) Immunoblot to detect recombinant proteins (immunoblotted for His tag) and HNK-1 carbohydrate modification. P21 brain lysate was used as a positive control for HNK-1 signal.

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Modification, Immunostaining, Slice Preparation, Labeling, Control, Immunoprecipitation, Western Blot, Recombinant, Positive Control

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan binds the Ig2 domain of NCAM, decreasing EphA3 binding. ( A ) Fc-pulldowns of the NCAM extracellular domain (NCAM-EC), truncation mutants of NCAM, or control Fc and recombinant neurocan. ( B ) Densitometry of ( A ) indicating the level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) for each construct (*p < 0.05 compared to NCAM-EC-Fc) (C) Fc-pulldowns of NCAM-EC-Fc or control Fc with mouse neurocan (lacking sushi domain) and full-length human neurocan. ( D ) Co-immunoprecipitation of WT NCAM-140 or mutants of NCAM and neurocan from transfected HEK293T cells. ( E ) Densitometry of ( D ). The amount of co-immunoprecipitated neurocan for each NCAM IP was normalized to control WT NCAM-bound neurocan (n = 3, t-test, *p < 0.05). ( F ) Immunoblot of untreated or chABC-treated neurocan protein probed for neurocan or C-4-S. ( G ) Co-immunoprecipitation of NCAM-140 and EphA3 from transfected HEK293 cells treated with no neurocan (control), neurocan, or chABC-treated neurocan. ( H ) Densitometry of ( G ). The amount of co-immunoprecipitated EphA3 for each NCAM IP was normalized to control NCAM-bound EphA3 (n = 3, t-test, *p < 0.05).

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Binding Assay, Control, Recombinant, Positive Control, Construct, Immunoprecipitation, Transfection, Western Blot

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan impairs ephrin-A5-mediated clustering of NCAM and EphA3 in cortical interneurons in culture. ( A ) Cortical neuron cultures were pretreated with no neurocan (control) or neurocan followed by preclustered Fc or ephrin-A5-Fc, and localization of endogenous NCAM (green) and EphA3 (red) was assessed in axons of GABA immunopositive axons by confocal microscopy. Scale bars = 5 μM. ( B ) Pearson’s Correlation Coefficients (R-Total) were generated for each condition using ImageJ co-localization software (n = 3, two-way ANOVA with Bonferonni post-hoc testing, *p < 0.05).

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Control, Confocal Microscopy, Generated, Software

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan decreases ephrin-A5-induced EphA3 autophosphorylation. ( A ) HEK293T cells transfected with NCAM and EphA3 were treated with preclustered control Fc or ephrin-A5-Fc, and EphA3 was immunoprecipitated. EphA3 autophosphorylation was assessed by immunoblotting with a phosphotyrosine antibody (PY99). Total levels of immunoprecipitated EphA3 were assessed by reprobing with EphA3 antibody. ( B ) Densitometry of ( A ). Graph indicates the ratio of phosphotyrosine to EphA3 values for each condition (n = 3, *p < 0.05). ( C ) Fc-pulldowns of control Fc, NCAM-EC-Fc, and ephrin-A5-Fc with recombinant neurocan. Level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) is indicated as a percentage under each lane. ( D ) Co-immunoprecipitation of NCAM (positive control) or EphA3 with neurocan from transfected HEK293T cells. Level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) is indicated as a percentage under each lane.

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Transfection, Control, Immunoprecipitation, Western Blot, Recombinant, Positive Control

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan inhibits ephrin-A5-induced growth cone collapse in GABAergic interneurons. ( A ) Representative spread and collapsed growth cones of GABA-immunostained interneurons in cortical neuron cultures. Scale bar = 5 μM. ( B ) The percentage of collapsed growth cones was determined for each condition (300 growth cones per condition, n = 3 experiments, 2-way ANOVA, Bonferonni post-hoc testing, ***p < 0.001).

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques:

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Model of inhibition of NCAM/EphA3 clustering and activation by neurocan. During postnatal remodeling of transient perisomatic synapses made by PV + interneurons onto pyramidal cell soma, ephrin-A5 dimers bind the ligand binding domains (LBD) of an EphA3 dimer. NCAM clusters the EphA3 receptors through binding of the NCAM Ig2 domain to the EphA3 CRD. EphA3 clustering activates tyrosine kinase signaling leading to synapse retraction. With further maturation, neurocan in PNNs engages the Ig2 domain of non-PSA NCAM, inhibiting EphA3 clustering and retraction of inhibitory perisomatic contacts. NCAM is shown in black, EphA3 is green, and ephrin-A5 is yellow. Neurocan core protein is depicted in purple with GAG chains in orange on a PNN scaffold (blue). P = phosphorylation. The illustration was created by modifying images purchased in the PPT Drawing Toolkits-BIOLOGY Bundle from Motifolio, Inc.

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Inhibition, Activation Assay, Ligand Binding Assay, Binding Assay, Phospho-proteomics

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: GAG-modified neurocan blocks chABC-induced decrease of perisomatic synaptic puncta in organotypic brain slices. ( A ) Immunostaining of neuronal soma (NeuN), a parvalbumin-positive interneuron (tdTomato), and a perineuronal net (WFA) in DIV14 organotypic brain slice culture. Scale bar = 10 μm. ( B ) WFA labeling of perineuronal nets in control penicillinase and chABC-treated brain slices. Scale bar = 30 μm. ( C ) Representative image of perisomatic synapses (tdTomato) in control penicillinase or chABC-treated slice cultures. Representative perisomatic puncta around a single soma are indicated with arrowheads. Scale bar = 10 μm. ( D ) Quantification of the mean number of perisomatic synaptic puncta per soma (n = 30 soma/condition, 3 animals per condition, t-test, *p < 0.05). ( E ) NCAM was immunoprecipitated from brain lysates, followed by immunoblotting with antibodies against neurocan, versican, or aggrecan/brevican (using an antibody raised against shared epitope). ( F ) Slices were treated with control penicillinase or chABC as in ( C ) followed by rescue with neurocan or tenascin-R. Quantification of the mean number of perisomatic synapses per soma was performed (>90 soma per mouse per condition, n = 3 mice, two-way ANOVA with Bonferonni post-hoc testing, *p < 0.05). ( G ) Immunoblot to detect recombinant proteins (immunoblotted for His tag) and HNK-1 carbohydrate modification. P21 brain lysate was used as a positive control for HNK-1 signal.

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Modification, Immunostaining, Slice Preparation, Labeling, Control, Immunoprecipitation, Western Blot, Recombinant, Positive Control

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan binds the Ig2 domain of NCAM, decreasing EphA3 binding. ( A ) Fc-pulldowns of the NCAM extracellular domain (NCAM-EC), truncation mutants of NCAM, or control Fc and recombinant neurocan. ( B ) Densitometry of ( A ) indicating the level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) for each construct (*p < 0.05 compared to NCAM-EC-Fc) (C) Fc-pulldowns of NCAM-EC-Fc or control Fc with mouse neurocan (lacking sushi domain) and full-length human neurocan. ( D ) Co-immunoprecipitation of WT NCAM-140 or mutants of NCAM and neurocan from transfected HEK293T cells. ( E ) Densitometry of ( D ). The amount of co-immunoprecipitated neurocan for each NCAM IP was normalized to control WT NCAM-bound neurocan (n = 3, t-test, *p < 0.05). ( F ) Immunoblot of untreated or chABC-treated neurocan protein probed for neurocan or C-4-S. ( G ) Co-immunoprecipitation of NCAM-140 and EphA3 from transfected HEK293 cells treated with no neurocan (control), neurocan, or chABC-treated neurocan. ( H ) Densitometry of ( G ). The amount of co-immunoprecipitated EphA3 for each NCAM IP was normalized to control NCAM-bound EphA3 (n = 3, t-test, *p < 0.05).

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Binding Assay, Control, Recombinant, Positive Control, Construct, Immunoprecipitation, Transfection, Western Blot

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan impairs ephrin-A5-mediated clustering of NCAM and EphA3 in cortical interneurons in culture. ( A ) Cortical neuron cultures were pretreated with no neurocan (control) or neurocan followed by preclustered Fc or ephrin-A5-Fc, and localization of endogenous NCAM (green) and EphA3 (red) was assessed in axons of GABA immunopositive axons by confocal microscopy. Scale bars = 5 μM. ( B ) Pearson’s Correlation Coefficients (R-Total) were generated for each condition using ImageJ co-localization software (n = 3, two-way ANOVA with Bonferonni post-hoc testing, *p < 0.05).

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Control, Confocal Microscopy, Generated, Software

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan decreases ephrin-A5-induced EphA3 autophosphorylation. ( A ) HEK293T cells transfected with NCAM and EphA3 were treated with preclustered control Fc or ephrin-A5-Fc, and EphA3 was immunoprecipitated. EphA3 autophosphorylation was assessed by immunoblotting with a phosphotyrosine antibody (PY99). Total levels of immunoprecipitated EphA3 were assessed by reprobing with EphA3 antibody. ( B ) Densitometry of ( A ). Graph indicates the ratio of phosphotyrosine to EphA3 values for each condition (n = 3, *p < 0.05). ( C ) Fc-pulldowns of control Fc, NCAM-EC-Fc, and ephrin-A5-Fc with recombinant neurocan. Level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) is indicated as a percentage under each lane. ( D ) Co-immunoprecipitation of NCAM (positive control) or EphA3 with neurocan from transfected HEK293T cells. Level of neurocan bound (relative to positive control NCAM-EC-Fc bound neurocan) is indicated as a percentage under each lane.

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Transfection, Control, Immunoprecipitation, Western Blot, Recombinant, Positive Control

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Neurocan inhibits ephrin-A5-induced growth cone collapse in GABAergic interneurons. ( A ) Representative spread and collapsed growth cones of GABA-immunostained interneurons in cortical neuron cultures. Scale bar = 5 μM. ( B ) The percentage of collapsed growth cones was determined for each condition (300 growth cones per condition, n = 3 experiments, 2-way ANOVA, Bonferonni post-hoc testing, ***p < 0.001).

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques:

Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Model of inhibition of NCAM/EphA3 clustering and activation by neurocan. During postnatal remodeling of transient perisomatic synapses made by PV + interneurons onto pyramidal cell soma, ephrin-A5 dimers bind the ligand binding domains (LBD) of an EphA3 dimer. NCAM clusters the EphA3 receptors through binding of the NCAM Ig2 domain to the EphA3 CRD. EphA3 clustering activates tyrosine kinase signaling leading to synapse retraction. With further maturation, neurocan in PNNs engages the Ig2 domain of non-PSA NCAM, inhibiting EphA3 clustering and retraction of inhibitory perisomatic contacts. NCAM is shown in black, EphA3 is green, and ephrin-A5 is yellow. Neurocan core protein is depicted in purple with GAG chains in orange on a PNN scaffold (blue). P = phosphorylation. The illustration was created by modifying images purchased in the PPT Drawing Toolkits-BIOLOGY Bundle from Motifolio, Inc.

Article Snippet: Recombinant ephrin-A5-Fc, human Fc, mouse neurocan, human neurocan, and human tenascin-R (R&D Systems) were also used.

Techniques: Inhibition, Activation Assay, Ligand Binding Assay, Binding Assay, Phospho-proteomics