RGMa aa 284–293 are critical for the interaction with neogenin.

<p>(A) Sequence alignment of the synthesized RGMa peptide. Peptides 1–6 were designed within aa 257–310, which is the region required for the interaction between RGMa and neogenin as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032791#pone-0032791-g001" target="_blank">Fig. 1</a>. (B) RGMa peptide 4 (aa 284–293) directly binds to neogenin's ECD. Relative binding of each RGMa peptide to ELISA plates coated with neogenin-ECD-Fc is shown. p75NTR-Fc was used as a control. Results (%) are shown as the mean ± SEM of 3 independent experiments (**<i>P</i><0.01, *<i>P</i><0.05).</p

RGMa peptide 4 (aa 284–293) inhibits RGMa-induced growth cone collapse.

<p>(A) Effects of RGMa peptide 4 on RGMa-induced growth cone collapse. Cortical neurons were treated with RGMa and/or RGMa peptides for 30 min. The results were quantified from 3 independent experiments, and the percentage of collapsed growth cones is shown (**<i>P</i><0.01). Pep4, peptide 4; PepA, Peptide A; PepB, peptide B. (B) Representative images of growth cones. Red: Phalloidin, Green: Tuj1. Scale bar: 10 µm.</p

The RGMa domain required for binding to neogenin is within aa 259–295.

<p>(A and C) Schematic representation of RGMa and its deletion mutants are shown with their domain structures. Arrowhead shows potential cleavage site. SP: signal peptide, vWD: von Willebrand factor type-D domain, HD: hydrophobic domain, GPI: GPI-anchor. (B, D, and E) Co-immunoprecipitation of full-length neogenin-VSV-G with the deletion mutants of RGMa-Myc. HEK293T cells were transiently transfected with the indicated plasmids. Cell lysates were immunoprecipitated with the anti-VSV-G antibody. The immunoprecipitates (IP) and cell lysates (Lysates) were analyzed by western blotting with anti-Myc and anti-VSV-G antibodies.</p

Neutrophils and macrophages in the CLNs after CCI.

<p>A: Dot plots of immune cells in the CLNs for live cell analysis. B: Representative cytometry data for neutrophils in the CLNs at the indicated days after CCI. C: Representative cytometry data for CD45⁺/CD11b⁺ macrophages in the CLNs at the indicated days after CCI. D: Graph illustrating quantitative data for neutrophils and macrophages in the CLNs after CCI. <i>n</i> = 3–4 for each experiment. Neutrophils: ** <i>p</i><0.01 at 1 and 14 dpi compared with no injury; macrophages: ** <i>p</i><0.01 at 7, 14, and 21 dpi compared with 1 dpi.</p

T cells and DCs in the cervical lymph nodes

<p>(<b>CLNs</b>) <b>after CCI.</b> A: Representative cytometry data for T cells in the CLNs at the indicated days after CCI. B: Graph illustrating quantitative data for T cells in the CLNs after CCI. C: Representative cytometry data for DCs in the CLNs at the indicated days after CCI. D: Graph illustrating quantitative data for DCs in the CLNs after CCI. (B, D) <i>n</i> = 3–4 for each experiment. T cells: * <i>p</i><0.05 at 28 dpi compared with 1 dpi; DCs: ** <i>p</i><0.01 at 3 dpi compared with no injury, * <i>p</i><0.05 at 7, 14, and 21 dpi compared with no injury.</p

Bimodal increase in the Iba-1-positive cells in the brain after CCI.

<p>(A–E) Brain sections immunostained for Iba-1 at the indicated days after CCI. The red signals show Iba-1-positive microglia/macrophages in sham-operated mouse brains (A) and in brains after CCI at 1 dpi (B), 7 dpi (C), 14 dpi (D), and 21 dpi (E). The small image on the right lower side of each large image is a magnified view of the square indicated. Scale bars: large image, 600 µm; small image 100 µm. (F) The graph shows the number of microglia/macrophages at the indicated days after CCI. Microglia/macrophages observed around the injured side significantly increased at 7 dpi and 21 dpi. Sham, sham-operated mice. <i>n</i> = 4 for each group. ** <i>p</i><0.01 at 7 and 21 dpi compared with sham controls.</p

Neutrophils and macrophages in the spleen after CCI.

<p>A: Dot plots of immune cells in the spleen for live cell analysis. B: Representative cytometry data for neutrophils in the spleen at the indicated days after CCI. C: Representative cytometry data for macrophages in the spleen at the indicated days after CCI. D: Graph illustrating quantitative data for neutrophils and macrophages in the CLNs after CCI. <i>n</i> = 3–5 for each experiment. Neutrophils: ** <i>p</i><0.01 at 28 dpi compared with no injury.</p

Changes in specific subsets of microglia in the injured brain.

<p>A: Representative cytometry data showing the purity of microglia isolated. Of the isolated cells, 91% were microglia. B: Representative cytometry data for CD86⁺/CD11b⁺ M1-like microglia in the brain at the indicated days after CCI. C: Representative cytometry data for CD206⁺/CD11b⁺ M2-like microglia in the brain at the indicated days after CCI. D: Graph illustrating quantitative data for CD86⁺/CD11b⁺ cells in the brain after CCI. CD86⁺/CD11b⁺ cells were higher in number than in the intact brain at 28 dpi. E: Graph illustrating quantitative data for CD206⁺/CD11b⁺ cells in the brain after CCI. CD206⁺/CD11b⁺ cells were higher in number at 7 dpi than in the intact brain, and decreased thereafter until 28 dpi. <i>n</i> = 3–5 for each group. * <i>p</i><0.05 compared with no injury.</p

Analysis of T cells and dendritic cells in the injured brain.

<p>A: Dot plots of isolated immune cells in the brain, gated for live cell analysis. B: Representative cytometry data for T cells (CD45⁺/CD3⁺ cells) in the injured brain at the indicated days after CCI. C: Representative cytometry data for CD45⁺/CD11c⁺ dendritic cells (DCs) in the injured brain after CCI. D: Graph illustrating quantitative data for accumulated T cells and DCs in the injured brain after CCI. <i>n</i> = 3–6 for each experiment. T cells: ** <i>p</i><0.01 at 1, 3, and 7 dpi compared with no injury, * <i>p</i><0.05 at 28 dpi compared with no injury; DCs: ** <i>p</i><0.01 at 3 and 7 dpi compared with no injury.</p

Analysis of neutrophils, macrophages, and microglia in the injured brain.

<p>A: Dot plots of isolated immune cells in the brain, gated for live cell analysis. B: Representative cytometry data for neutrophils (CD45⁺/Gr-1⁺ cells) in the injured brain at the indicated days after CCI. C: Representative cytometry data for macrophages (CD45^high/CD11b⁺ cells) and microglia (CD45^low/CD11b⁺ cells) in the injured brain at the indicated days after CCI. D: Graph illustrating quantitative data for accumulated neutrophils, macrophages, and microglia in the injured brain up to 28 d after CCI. All the values are presented in terms of mean ± standard error of mean (<i>n</i> = 3–6). Macrophages/neutrophils: ** <i>p</i><0.01 at 1, 3, and 14 dpi compared with no injury; microglia: ** <i>p</i><0.01 at 14 and 28 dpi compared with no injury, * <i>p</i><0.05 at 14 dpi compared with 1 dpi.</p