Journal: Frontiers in Neuroscience

Article Title: Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse

doi: 10.3389/fnins.2023.1100121

Figure Lengend Snippet: Caspr2, TAG-1, and Cntnap2 mRNA levels in mouse brains during development. Representative immunoblots showing Caspr2, TAG-1, and GAPDH levels in brain lysates of wild-type (WT), HET, and KO mice at different developmental stages and at adulthood (A) . Levels of Caspr2 normalized to GAPDH levels and relative to mean WT (in %) (B) . Levels of Cntnap2 mRNAs normalized to the Psap gene and relative to mean WT (ratio) in brain of WT, HET, and KO mice at E17.5, P7, P10, and P30 (C) . Levels of TAG-1 normalized to GAPDH levels and relative to mean WT (in %) (D) . (B–D) Six animals/genotype/age. Statistical tests: (B) Mann–Whitney test to compare Caspr2 levels in HET mice at stages E17.5, P2, P7, P10, P15, or P30, to Caspr2 level in HET mice at P90. (C) Unpaired t -test to compare Cntnap2 mRNA levels in HET mice at stages E17.5, P7, or P10, to Cntnap2 mRNA level in HET mice at P30. (D) One-way ANOVA test or Kruskal–Wallis test to compare the three genotypes at different ages; * P < 0.05, ** P < 0.01, **** P < 0.0001; ns, non-significant.

Article Snippet: Briefly, slides were treated with 0.1 M glycine for 30 min at RT or with methanol 50%/acetone 50% for 20 min at −20°C, pre-incubated for 1 h at room temperature in PS2, before incubation with primary antibodies diluted in PS2 overnight at 4°C (K v 1.2 α subunit, mouse clone K67/25 #75-075, NeuroMab, 1:400; F3, goat #AF904, R&D Systems, 1:400; NrCAM, rabbit #ab24344, Abcam, Cambridge, UK, 1:300; TAG-1, goat #AF4439, R&D Systems, 1:500; Caspr (L51, 1:500) and Caspr2 (191, 1:400) previously described) ( ; ).

Techniques: Western Blot, MANN-WHITNEY

Journal: Frontiers in Neuroscience

Article Title: Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse

doi: 10.3389/fnins.2023.1100121

Figure Lengend Snippet: Myelinated fiber abnormalities in sciatic nerves of adult mice. Electron micrographs of transversal sections of the sciatic nerve of wild-type (WT), HET, and KO adult mice, showing a single myelinated fiber for each genotype (A) . Axonal diameters of myelinated fibers (B) . Scatter plot graph displaying G-ratios of individual myelinated axons as a function of the respective axon diameters, and the linear regression of the G-ratio measurements for each genotype (C) . Representative confocal images of immunostainings of sciatic nerves fibers from WT, HET, and KO adult mice, for nodal (NrCAM), paranodal (Caspr, F3), and juxtaparanodal (Caspr2, TAG-1, Kv1.2) proteins (D) . Length of the nodes measured on NrCAM immunostainings (E) . Scatter plot graph displaying length of individual node as a function of the respective nodal diameters, and the linear regression of the node length measurements for each genotype (F) . Number of slips per 100 steps made by WT, HET, and KO adult males during the grid-walking test (G) . (B,C) 141 myelinated fibers/genotype, 4 mice/genotype, 47 myelinated fibers/mouse, (E,F) 243 nodes/genotype, 3 mice/genotype, 81 nodes/mouse, (G) 8–9 mice/genotype. Statistical tests: (B,E) Kruskal–Wallis test to compare the three genotypes; (C) Linear regression (WT R 2 = 0.2093, HET R 2 = 0.1143, KO R 2 = 0.2022) for pairwise comparisons, HET vs. WT elevation P < 0.0001, HET vs. KO slope P = 0.019; (F) Linear regression (WT R 2 = 0.2295, HET R 2 = 0.1368, KO R 2 = 0.1935) for pairwise comparisons, HET vs. WT elevation P = 0.0052, KO vs. WT slope P = 0.0315, HET vs. KO elevation P < 0.0001; (G) one-way ANOVA test to compare the three genotypes; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Bar scales, 10 μm (E) .

Article Snippet: Briefly, slides were treated with 0.1 M glycine for 30 min at RT or with methanol 50%/acetone 50% for 20 min at −20°C, pre-incubated for 1 h at room temperature in PS2, before incubation with primary antibodies diluted in PS2 overnight at 4°C (K v 1.2 α subunit, mouse clone K67/25 #75-075, NeuroMab, 1:400; F3, goat #AF904, R&D Systems, 1:400; NrCAM, rabbit #ab24344, Abcam, Cambridge, UK, 1:300; TAG-1, goat #AF4439, R&D Systems, 1:500; Caspr (L51, 1:500) and Caspr2 (191, 1:400) previously described) ( ; ).

Techniques:

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Schematic representation of LRP-1-derived minireceptors carrying no-ligand-binding cluster (SPCT), extracellular binding-domain II (DII) or extracellular binding-domain IV (DIV). Each construct contains a HA tag at the amino-terminus of the α-chain. B. Transfected CHO cells stably express HA-tagged SPCT (SPCT), HA-tagged mini LRP-II (DII), or HA-tagged mini LRP-IV (DIV). Nontransfected cells served as control (CTRL). Biotinylation of cell-surface proteins was performed, followed by an immunoblot (IB) analysis using anti-HA tag. Bands correspond to the expected molecular weights of SPCT (106 kDa; arrowhead), DII (153 kDa; star), and DIV (164 kDa; double star). C. CHO cells overexpressing HA-tagged LRP-1-derived minireceptors (SPCT, DII, DIV) or not (CTRL) were transiently transfected with RFP-tagged TIMP-1 for 24 hours. Cell-surface proteins were subjected to immunoprecipitation (IP) assay with either anti-HA tag (left panel) or an anti-RFP tag (right panel). Then, immunoblot (IB) analysis was conducted using both anti-LRP-1 β-chain (5A6) and anti-RFP tag. D. Representative sensorgrams for TIMP-1 interacting with DII (left panel) and DIV (right panel). A set of concentrations (5–80 nM) of TIMP-1 or EGF was sequentially injected over immobilized Fc-DII and Fc-DIV. The solid black lines represent the specific binding of TIMP-1 obtained after double-subtraction of the signal obtained on the control flow cell and a blank run. The dotted black lines represent the fit of the data with a kinetic titration 1∶1 interaction model. The grey lines represent the specific binding of EGF obtained after double-subtraction of the signal obtained on the control flow cell and a blank run. Arrows indicate the beginning of each injection. The data illustrated are representative of three independent experiments. E. Binding and internalization of exogenous fluorescent TIMP-1 (fluo-TIMP-1) by CHO cells overexpressing minireceptor SPCT (left), DII (middle) or DIV (right). Binding was assessed by incubating fluo-TIMP-1 (10 nM) at 4°C for 2 hours. Cells were then transferred to 37°C for additional 2 h to allow internalization. All incubations were performed with or without RAP (500 nM), an antagonist of LRP-1-mediated binding and consequently, endocytosis. Fluorescence intensity was quantified by spectrophotometry and expressed as arbitrary units (A.U.). Values below 10 A.U. are considered to be nonspecific. Images in B–D are representative of results obtained in 3 independent experiments. Values in E represent the means ± s.e.m. of 3 independent experiments. NS, not significant; * p <0.05, as compared to untreated cells.

Article Snippet: Recombinant human LRP-1 Fc-tagged mini-domains II and IV (termed respectively Fc-DII and Fc-DIV) were purchased from R&D Systems.

Techniques: Derivative Assay, Ligand Binding Assay, Binding Assay, Construct, Transfection, Stable Transfection, Western Blot, Immunoprecipitation, Injection, Titration, Fluorescence, Spectrophotometry

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Cortical neurons from mouse embryos were plated onto poly-L-lysine-coated coverslips for 24 h at 37°C, fixed, washed and stained with anti-LRP1 antibody (Alexa Fluor 488, green) and anti-TIMP-1 antibody (Alexa Fluor 568, red) before confocal microscopy analysis. Nuclei were counterstained with DAPI (blue) and appropriate secondary antibody controls were performed. LRP-1 labeling (left), TIMP-1 labeling (middle), and a merged image (right) are shown. B. Biotinylation of cell-surface proteins was conducted at 4°C from cortical neurons previously treated for 24 h with or without RAP (500 nM). Proteins were affinity precipitated with avidin-agarose beads, then LRP-1-containing complexes were immunoprecipitated by either anti-LRP-1 β-chain (LRP-1 β; left panel) or anti-LRP-1 α-chain (LRP-1 α; middle panel) and analyzed by western-blot using anti-LRP-1 β-chain (5A6), anti-LRP-1 α-chain (8G1) and anti-TIMP-1 antibodies. Nonspecific IgGs were used as a negative control of immunoprecipitation. The presence of TIMP-1 in immunocomplexes was quantified by densitometric analysis relative to immunoprecipitated LRP-1-α-chain (histogram, right panel). C. Binding and internalization of exogenous fluorescent TIMP-1 (fluo-TIMP-1) by cortical neurons. Binding was determined by incubating fluo-TIMP-1 at 4°C for 2 h. After extensive washes, part of the cells was used to quantify total binding. The other part was incubated at 37°C for an additional 1 h to permit endocytosis. Experiments were carried out with or without RAP (500 nM). Fluorescence intensity was quantified by spectrophotometry and expressed as arbitrary units (A.U.). Values below 10 A.U. are considered to be nonspecific. Images in A and B are representative of results obtained in 3 independent experiments. Values in B and C represent the means ± s.e.m. of 3 independent experiments. NS, not significant; ** p <0.01, as compared to untreated cells. Scale bar: 5 µm.

Article Snippet: Recombinant human LRP-1 Fc-tagged mini-domains II and IV (termed respectively Fc-DII and Fc-DIV) were purchased from R&D Systems.

Techniques: Staining, Confocal Microscopy, Labeling, Avidin-Biotin Assay, Immunoprecipitation, Western Blot, Negative Control, Binding Assay, Incubation, Fluorescence, Spectrophotometry

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Cortical neurons from mouse embryos were cultured for 24-L-lysine-coated coverslips and then treated for 30 min with TIMP-1 (10 nM), RAP (500 nM), blocking LRP-1 polyclonal antibodies (R2629) or a combination of TIMP-1+RAP and TIMP-1+R2629. Untreated cells served as control (CTRL). Cells were labeled with anti-βIII-tubulin monoclonal antibody and observed under confocal microscopy. B. Quantification of neurite mean length per cell was performed using the ImageJ plugin NeuronJ and expressed as percent of untreated neurons (CTRL). Images in A are representative of results obtained in 3 independent experiments. Values in B represent the means ± s.e.m. of 3 independent experiments. NS, not significant; ** p <0.01. Scale bar: 10 µm.

Article Snippet: Recombinant human LRP-1 Fc-tagged mini-domains II and IV (termed respectively Fc-DII and Fc-DIV) were purchased from R&D Systems.

Techniques: Cell Culture, Blocking Assay, Labeling, Confocal Microscopy

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Cortical neurons from mouse embryos were treated after 24-L-lysine-coated coverslips for 30 min with TIMP-1 (10 nM), RAP (500 nM) or blocking LRP-1 polyclonal antibodies (R2629) or a combination of TIMP-1+RAP and TIMP-1+R2629. Untreated cells served as a control (CTRL). Neurons were incubated with Alexa Fluor 568-phalloidin to label F-actin structures and analyzed by confocal microscopy. B. 3D-quantification of growth cone volume was performed using the AMIRA software and expressed as percent of untreated neurons (CTRL). Images in A are representative of results obtained in 3 independent experiments. Values in B represent the means ± s.e.m. of 3 independent experiments. NS, not significant; ** p <0.01. Scale bar: 5 µm.

Article Snippet: Recombinant human LRP-1 Fc-tagged mini-domains II and IV (termed respectively Fc-DII and Fc-DIV) were purchased from R&D Systems.

Techniques: Blocking Assay, Incubation, Confocal Microscopy, Software

Journal: PLoS ONE

Article Title: Low-Density Lipoprotein Receptor-Related Protein-1 Mediates Endocytic Clearance of Tissue Inhibitor of Metalloproteinases-1 and Promotes Its Cytokine-Like Activities

doi: 10.1371/journal.pone.0103839

Figure Lengend Snippet: A. Cortical neurons from mouse embryos were allowed to grow during 24-L-lysine-coated coverslips, and treated for 30 min with FLAG-TIMP-1 (10 nM) or FLAG-T2G (10 nM). Neurons were then stained with anti-LRP-1 antibody (Alexa Fluor 568, red) or anti-FLAG antibody (Alexa Fluor 488, green) and analyzed by confocal microscopy. Nuclei were counterstained with DAPI (blue). Images were treated with the AMIRA sofware. Fluorescent signals corresponding to LRP-1, FLAG and colocalization were shown by red (left), green (middle) and cyan (right) labeling. B–C. Neurons were treated as indicated in A , in the absence or presence of RAP. B. Neurites were labeled with anti-βIII-tubulin antibody and observed under confocal microscopy. The neurite mean length per cell was determined using the ImageJ plugin NeuronJ and expressed as percent of untreated neurons (CTRL). C. Actin-rich growth cones were visualized with Alexa Fluor 568-phalloidin, observed under confocal microscopy and quantified using the AMIRA software (right panel). Images in A are representative of results obtained in 3 independent experiments. Values in B and C represent the mean ± s.e.m. of 3 independent experiments. NS, not significant; ** p <0.01. Scale bar: 5 µm.

Article Snippet: Recombinant human LRP-1 Fc-tagged mini-domains II and IV (termed respectively Fc-DII and Fc-DIV) were purchased from R&D Systems.

Techniques: Staining, Confocal Microscopy, Labeling, Software

Journal: PLoS ONE

Article Title: Dkk1 Stabilizes Wnt Co-Receptor LRP6: Implication for Wnt Ligand-Induced LRP6 Down-Regulation

doi: 10.1371/journal.pone.0011014

Figure Lengend Snippet: (A) HEK293 cells in 6 well plates were transiently transfected with 0.8 µg of Myc-tagged human LRP6 plasmid. Twenty four hours following transfection, cells were cultured with Dkk1 CM (+) or vector-transfected control CM (−) for additional 24 h. The level of LRP6 was then examined by Western blotting using the anti-Myc antibody. Similarly, HT1080 cells stably transduced with HA-tagged LRP6 in 6-well plates were cultured with Dkk1 CM or control CM for 24 h, and the level of LRP6 was then examined by Western blotting with the anti-HA antibody. (B) HT1080 cells stably transduced with HA-tagged LRP6 were cultured with Dkk1 CM or control CM for 4, 8, or 16 h. The level of LRP6 was then examined by Western blotting with the anti-HA antibody. (C) HT1080 cells stably transduced with HA-tagged LRP6 in 6 well plates were cultured with recombinant Dkk1 protein (1 µg/ml) or recombinant sFRP1 (1 µg/ml) for 24 h. The level of LRP6 was then examined by Western blotting with the anti-HA antibody. Samples were also probed with anti-actin antibody to verify equal loading.

Article Snippet: Human recombinant Dkk1 and sFRP1 proteins were obtained from R&D Systems.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Western Blot, Stable Transfection, Transduction, Recombinant

Journal: Molecular Vision

Article Title: Chordin-like 1, a bone morphogenetic protein-4 antagonist, is upregulated by hypoxia in human retinal pericytes and plays a role in regulating angiogenesis

doi:

Figure Lengend Snippet: Validation of three genes differentially expressed in human retinal pericytes (hRPC) in response to hypoxia. The upregulation of a selection of genes was validated with real time PCR, using a PerkinElmer 7700 analyzer, on cDNA generated from human retinal pericytes exposed to increasing periods of hypoxia (0, 6, 24, and 48 h). All results were normalized to 18S rRNA, using a pre-developed assay reagent. Data are expressed as mean relative quantity of mRNA, relative to control, for three independent experiments ±standard error of measurement for ( A ) CHL-1 mRNA, ( B ) VEGF mRNA, and ( C ) Cox 2 mRNA. Data are expressed as mean±SEM values. The asterisk indicates a significance at p<0.05 and the double asterisk indicates a significance at p<0.001.

Article Snippet: Whole cell extracts were examined by western blotting for V5 tagged CHL-1 expression using an anti-V5 antibody (Invitrogen; ) Whole cell extracts from Cos7 cells transfected with empty pcDNA6/V5His (E), or with the expression vector pcDNA6 CHL-1/V5His (CHL-1) expressing V5His tagged CHL-1, were incubated with 250 ng rhBMP-4 (R&D Systems) and 100 ml NiNTA magnetic beads at 4 °C overnight with rotation.

Techniques: Selection, Real-time Polymerase Chain Reaction, Generated

Journal: Molecular Vision

Article Title: Chordin-like 1, a bone morphogenetic protein-4 antagonist, is upregulated by hypoxia in human retinal pericytes and plays a role in regulating angiogenesis

doi:

Figure Lengend Snippet: HIF-1α drives expression of chordin-like 1 in retinal pericytes exposed to hypoxia. A : western blot analysis of nuclear extracts generated from human retinal pericytes exposed to increasing periods of hypoxia (0, 6, 24, and 48 h) for HIF-1α shows upregulation of the protein by 6 h. B : Transfection of human retinal pericytes maintained in normoxia with expression vectors for HIF-1 α, C is control/empty vector, WT is wild type vector, WT-HIF-1 α, DM is double mutant vector, DM-HIF-1 α, using the transfection reagent Fugene6, induces expression of many of the genes upregulated in response to hypoxia, as measured by RT–PCR. 18S PCR is shown as a loading control and western blot analysis confirmed expression from each of the HIF-1α expressing plasmids. C : Induction of CHL-1 mRNA in response to HIF-1α overexpression was quantitated by real time PCR. CHL-1 levels were normalized to 18S rRNA, using a pre-developed assay reagent. Data are expressed as mean relative quantity of mRNA, to control, for three independent experiments ±standard error of measurement. D : HeLa cells were transfected, using the transfection reagent Fugene6, with the CHL-1 promoter or a luciferase reporter construct containing four HIF-1α responsive elements (HRE), alone (control), cotransfected with the HIF-1α expression vector DM-HIF-1α, or alone and subsequent exposure to hypoxia for 24 h (1% O 2 ). Cotransfection with DM-HIF-1α as well as exposure to hypoxia induced activation of the CHL-1 promoter and the HRE construct. Data are expressed as mean±SEM values. The asterisk indicates a significance at p<0.05 and the double asterisk indicates a significance at p<0.001.

Article Snippet: Whole cell extracts were examined by western blotting for V5 tagged CHL-1 expression using an anti-V5 antibody (Invitrogen; ) Whole cell extracts from Cos7 cells transfected with empty pcDNA6/V5His (E), or with the expression vector pcDNA6 CHL-1/V5His (CHL-1) expressing V5His tagged CHL-1, were incubated with 250 ng rhBMP-4 (R&D Systems) and 100 ml NiNTA magnetic beads at 4 °C overnight with rotation.

Techniques: Expressing, Western Blot, Generated, Transfection, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Over Expression, Real-time Polymerase Chain Reaction, Luciferase, Construct, Cotransfection, Activation Assay

Journal: Molecular Vision

Article Title: Chordin-like 1, a bone morphogenetic protein-4 antagonist, is upregulated by hypoxia in human retinal pericytes and plays a role in regulating angiogenesis

doi:

Figure Lengend Snippet: Chordin-like 1 expressed in human retinal pericytes is secreted and binds to bone morphogenetic protein-4. A : Conditioned media from HRPC exposed to 1% O 2 for 24 and 48 h was examined by western blot analysis for secretion of CHL-1, using an anti-CHL-1 antibody. B and C : BMP-2 and BMP-4 expression in HRPC was examined in cells cultured in normoxia (N) and hypoxia (H) by RT–PCR (B) and secreted BMP2 and BMP-4 were detected in conditioned media from HRPC exposed to 1% O 2 for 24 and 48 h (C). D: Transfection of the expression vector pcDNA6/CHL-1 V5-His into Cos7 cells, using the transfection reagent Fugene 6, resulted in expression of an approximately 60 kDa protein, which was detectable using an anti-V5 antibody. Cells were transfected with either an empty vector (E), pcDNA6/V5-His C, or a V5 tagged CHL-1 expressing vector (CHL-1), pcDNA6/CHL-1 V5-His. E: Whole cell extracts from Cos7 cells were transfected, using the transfection reagent Fugene 6, with empty pcDNA6/V5His (E), or with the expression vector pcDNA6 CHL-1/V5His (CHL-1) expressing V5His tagged CHL-1, were incubated with 250 ng rhBMP-4 and 100 ml NiNTA magnetic beads at 4 °C overnight. The complexes were washed, the beads and examined by western blotting for the presence of CHL-1, using anti-V5 antibody, and BMP-4, using an anti-BMP-4 antibody.

Article Snippet: Whole cell extracts were examined by western blotting for V5 tagged CHL-1 expression using an anti-V5 antibody (Invitrogen; ) Whole cell extracts from Cos7 cells transfected with empty pcDNA6/V5His (E), or with the expression vector pcDNA6 CHL-1/V5His (CHL-1) expressing V5His tagged CHL-1, were incubated with 250 ng rhBMP-4 (R&D Systems) and 100 ml NiNTA magnetic beads at 4 °C overnight with rotation.

Techniques: Western Blot, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Incubation, Magnetic Beads

Journal: Molecular Vision

Article Title: Chordin-like 1, a bone morphogenetic protein-4 antagonist, is upregulated by hypoxia in human retinal pericytes and plays a role in regulating angiogenesis

doi:

Figure Lengend Snippet: Chordin-like 1 modulates the antiangiogenic effect of bone morphogenetic protein-4. Human umbilical vascular endothelial cells(HUVECs) and human diploid fibroblasts were obtained (day 1) as cocultures in 24 well plates. Medium, with treatments or vehicle, was replenished on days 1, 4, 7, and 9. The assay was treated with VEGF, Suramin, recombinant human BMP-2, BMP-4, and CHL-1. Tubule formation was examined at day 11. Cells were fixed, quantitated, and visualized using a combined ELISA and histology kit. A : VEGF (2 ng/ml) and Suramin (20 mM) were used as positive and negative angiogenesis controls, respectively. B: Cells were treated with rhBMP-2 and rhBMP-4. BMP-4 significantly inhibited angiogenesis at 10 ng/ml. C : CHL-1 inhibited BMP-4; CHL-1 alone had no significant effect on angiogenesis, however it inhibited BMP-4s anti-angiogenic effects. Images A-C are shown at magnification 10X. Representative images are shown in A-C . D : Angiogenesis was quantitated by using anti-CD31 antibody coupled to a soluble substrate, ρ-nitrophenol phosphate (ρ-NPP), which permits quantitation by an optical density measurement. The asterisk indicates a significance at p<0.05 and the double asterisk indicates a significance at p<0.001.

Article Snippet: Whole cell extracts were examined by western blotting for V5 tagged CHL-1 expression using an anti-V5 antibody (Invitrogen; ) Whole cell extracts from Cos7 cells transfected with empty pcDNA6/V5His (E), or with the expression vector pcDNA6 CHL-1/V5His (CHL-1) expressing V5His tagged CHL-1, were incubated with 250 ng rhBMP-4 (R&D Systems) and 100 ml NiNTA magnetic beads at 4 °C overnight with rotation.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Quantitation Assay