sheep anti rgmb  (R&D Systems)

Journal: Cell

Article Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

doi: 10.1016/j.cell.2021.02.045

Figure Lengend Snippet:

Article Snippet: The antibodies we used were goat anti-NEO1 (1:200, AF1079; R&D systems) and sheep anti-RGMB (1:200, AF3597; R&D systems).

Techniques: Produced, Virus, Recombinant, Modification, Protease Inhibitor, Immunoprecipitation, Staining, Infection, Plasmid Preparation, Sequencing, Purification, Transformation Assay, Proximity Ligation Assay, In Situ, Imaging, Structural Proteomics, Software, Western Blot

Journal: Cell

Article Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

doi: 10.1016/j.cell.2021.02.045

Figure Lengend Snippet: Identification of the minimal NEO1-NET1 interaction region, related to Figure 1 (A, B) SPR equilibrium binding experiments of different NET1 and NEO1 constructs. Graphs show a plot of the equilibrium binding response against used NEO1 construct concentrations (left panels: full-length NEO1 ectodomain (eNEO1), right panels: NEO1 FN type III domains 4 to 6 (NEO1 FN456 ). Ligands immobilized on SPR sensor chip: A , full-length NET1; B , NET1 ΔNTR . (C) Immunofluorescence staining of FLAG-tagged full-length human DCC (DCC FL ) and mouse NEO1 (NEO1 FL ) overexpressed in COS-7 cells (green). Left panel: bound NET1 ΔNTR is stained via a Rho ID4 tag (red); right panel: transfected cells were incubated with buffer only as a negative control and stained as in the left panel. (D) Western blot of COS-7 cells transfected with the indicated plasmids used in C . α-tubulin serves as a loading control. (E, F) Proximity ligation assay (PLA) to test for simultaneous binding of NET1 and RGMB to NEO1. (E) COS-7 cells were transfected with a NEO1-mVenus fusion protein or the corresponding empty vector, and with full-length RGMB (wild type or RGMB-A186R). Transfected cells were incubated with NET1 ΔNTR before performing the PLA assay. PLA signals are shown in red and NEO1-mVenus transfected cells in green with nuclei in blue. (F) PLA signals were quantified and values from 3 individual experiments were plotted. A two-tailed, unpaired t test showed the statistical significance as p = 0.0107.

Article Snippet: The rest of the lysate was split into half, one of these halves was incubated with 0.5 μg of non-specific sheep IgGs (R&D Systems 5-001-A) and the other half with 0.5 μg of sheep anti-RGMB antibody (R&D AF3597), followed by incubation with 5 μl of magnetic beads (Dynabeads Protein G, Thermofisher) for 1hr at 4°C on a rotating wheel.

Techniques: Binding Assay, Construct, Immunofluorescence, Staining, Transfection, Incubation, Negative Control, Western Blot, Proximity Ligation Assay, Plasmid Preparation, Two Tailed Test

Journal: Cell

Article Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

doi: 10.1016/j.cell.2021.02.045

Figure Lengend Snippet: NET1 and RGMB can simultaneously bind NEO1 and form a ternary complex (A) Schematics of NEO1, NET1, and RGMB. SP, signal peptide; TM, transmembrane helix; IG, immunoglobulin-like domain; FN, fibronectin type III domain; CD, cytoplasmic domain; LN, laminin domain; LE, laminin epidermal growth factor-like repeats; LC, netrin (NTR) domain; N-RGM, RGM N-terminal domain identified to bind to BMP ligands ( Healey et al., 2015 ); vWfD, von Willebrand factor D-like domain; GPI, glycosylphosphatidylinositol anchor. (B and C) Proximity ligation assays (PLA) were performed to test for simultaneous binding of NET1 and RGMB to NEO1. (B) Cos-7 cells were either transfected with a NEO1-mVenus fusion protein or empty vector. Cells were incubated with NET1 ΔNTR and RGMB ECD (wild type or RGMB ECD -A186R). NEO1-mVenus positive cells are shown in green, nuclei are stained with DAPI and PLA signals in red. (C) Number of PLA signals per NEO1-mVenus positive cells. Individual values are plotted from 4 independent experiments. Statistical significance was determined using a two-tailed, unpaired t test with p < 0.0001. (D) Ribbon representation of the NEO1-NET1-RGMB protomer observed in the 3.25 Å resolution crystal structure, with NEO1 FN456 in red, NET1 ΔNTR in blue and RGMB CORE in yellow. A schematic is shown. See also Figure S1 .

Article Snippet: The rest of the lysate was split into half, one of these halves was incubated with 0.5 μg of non-specific sheep IgGs (R&D Systems 5-001-A) and the other half with 0.5 μg of sheep anti-RGMB antibody (R&D AF3597), followed by incubation with 5 μl of magnetic beads (Dynabeads Protein G, Thermofisher) for 1hr at 4°C on a rotating wheel.

Techniques: Ligation, Binding Assay, Transfection, Plasmid Preparation, Incubation, Staining, Two Tailed Test

Journal: Cell

Article Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

doi: 10.1016/j.cell.2021.02.045

Figure Lengend Snippet: Interface analysis of the ternary NEO1-NET1-RGMB super-complex (A) Close-up views of the observed NET1-NEO1 interfaces (right: interface 1, left: interface 2). Residues are displayed in stick representation and labelled according to domain color-coding. A Ca 2+ ion bound to NET1 LN (grey sphere) and hydrogen bonds (dashed black lines) are displayed. Mutated residues are in bold and underlined. (B) SPR equilibrium binding curves for the NET1-NEO1 interaction. A schematic of the experiment and the calculated K d values are shown. (C) AUC analysis of the NEO1 FN456 -NET1 ΔNTR -RGMB ECD complex, using NET1 ΔNTR WT and mutants. Both NET1 interface-1 and -2 mutants abolish the 3:3:3 stoichiometry of the NEO1-NET1-RGMB super-complex. (D) Overlapping expression of NET1 RNA (in situ hybridization), and NEO1 and RGMB protein (immunohistochemistry) in consecutive coronal sections of E16 mouse striatum. Boxed area is shown at higher magnification for NEO1 and RGMB. Scale bar, 100 μm. (E) RGMB immunoprecipitation (IP) from adult mouse cortex was followed by immunoblotting. Input samples (lane 1), IP using control non-specific IgGs (cntrl) (lane 2), and anti-RGMB IP (lane 3). NEO1 and NET1 co-IP with RGMB from adult mouse brain lysates. (F and G) Functional analysis of the effect of NET1 on RGMA-mediated growth cone collapse. (F) Representative examples of growth cones from mouse P0 cortical neurons. Neurons were stained with the microtubule marker Tuj1 (green) and F-actin marker phalloidin (red). Scale bar, 10 μm. (G) Quantification of growth cone collapse. Growth cones were treated with control or RGMA alone and in combination with different NET1 variants. Proportions of collapsed growth cones relative to control are displayed. n = 3 experiments, one-way ANOVA followed by Tukey’s multiple comparison test. ∗ p < 0.05. Data are shown as means ± SEM. (H–J) Comparison of binary NEO1-RGM (PDB ID 4BQ6 [ Bell et al., 2013 ]) and the ternary NEO1-NET1-RGMB complexes shown as ribbons. The ternary NEO1-NET1-RGMB protomer complex architecture (I) clashes with the NEO1-RGM dimer-of-dimers signaling conformation (H) when superimposed on NEO1 (marked with an asterisk) (J). See also Figure S3 , Figure S4 , Figure S5 .

Article Snippet: The rest of the lysate was split into half, one of these halves was incubated with 0.5 μg of non-specific sheep IgGs (R&D Systems 5-001-A) and the other half with 0.5 μg of sheep anti-RGMB antibody (R&D AF3597), followed by incubation with 5 μl of magnetic beads (Dynabeads Protein G, Thermofisher) for 1hr at 4°C on a rotating wheel.

Techniques: Binding Assay, Expressing, RNA In Situ Hybridization, Immunohistochemistry, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Functional Assay, Staining, Marker

Journal: Cell

Article Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

doi: 10.1016/j.cell.2021.02.045

Figure Lengend Snippet:

Article Snippet: The rest of the lysate was split into half, one of these halves was incubated with 0.5 μg of non-specific sheep IgGs (R&D Systems 5-001-A) and the other half with 0.5 μg of sheep anti-RGMB antibody (R&D AF3597), followed by incubation with 5 μl of magnetic beads (Dynabeads Protein G, Thermofisher) for 1hr at 4°C on a rotating wheel.

Techniques: Produced, Recombinant, Modification, Protease Inhibitor, Immunoprecipitation, Staining, Infection, Plasmid Preparation, Sequencing, Purification, Transformation Assay, Proximity Ligation Assay, In Situ, Imaging, Software, Western Blot