Journal: The Journal of Biological Chemistry

Article Title: Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth *

doi: 10.1074/jbc.M112.389916

Figure Lengend Snippet: Secreted Olfm1-AP binds to NgR1 on the surface of COS7 cells. A, schematic diagram showing three different isoforms (AMZ, BMZ, and BMY), and a deletion mutant (AMZdelEx4) of the Olfm1 protein, their domain structures, and possible modification sites. The alternative N-terminal sequences, A and B, that include signal peptides are shown in yellow and orange, the central M part of Olfm1 is common for all isoforms and is shown in green. The olfactomedin domain is marked in pink. B, Olfm1-AP binds to NgR1 but not to other tested receptor and membrane proteins on the surface of COS7 cells. COS7 cells transfected with receptor-expressing constructs are indicated in each panel. Two days later, cells were incubated with 10 nm Olfm1 (AMZ)-AP and stained for bound AP. Scale bar = 500 μm. C, Olfm1 (AMZ)-AP (10 nm) binds to NgR1 more strongly than to NgR2 or NgR3, and this binding was inhibited by soluble NgR1 protein (NgRsol) (5 nm). Scale bar = 500 μm. D, Scatchard plot of Olfm1 (AMZ)-AP binding to NgR1. These experiments were repeated four times, and results of typical experiment are shown. E, binding of different isoforms of Olfm1-AP, a deletion mutant of Olfm1-AP and MAG-AP, to COS7 cells transfected with NgR1. The AP activities in all CM were adjusted to the same concentration before adding to the NgR1 cells. Note that mutated Olfm1 with a deletion in the central region (delEx4) exhibits reduced activity. Scale bar = 500 μm. F, quantification of the results shown in E. G, binding of Olfm1-AP to COS7 cells transfected with full-length and deletion mutants of NgR1. The schematic diagram shows the main structural features of the NgR1 protein. The N- and C-terminal cysteine-rich cupping domains are shown as a blue rectangle and oval, respectively. Eight leucine-rich repeats are marked by transverse black lines. NT, N-terminal capping domain; CT, C-terminal capping domain. Scale bar = 500 μm. H, binding of Olfm1-AP to the sections from mouse P3 olfactory bulbs. Fresh sections (200 μm) were incubated with AP (left panel) or Olfm1-AP (center and right panels) together with normal rabbit IgG (left and center panels) or anti-NgR1 polyclonal IgG (right panel) at the concentration of 5 ng IgG/μl. The arrowheads show positive cells in different cell layers. EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer. Scale bar = 100 μm.

Article Snippet: The primary antibodies used were anti-Olfm1 (1:100 dilution), anti-NgR1 (1:50 dilution, Alomone Laboratory), and anti-EGFP (1:2,000 dilution, Aves Labs, Inc.).

Techniques: Mutagenesis, Modification, Transfection, Expressing, Construct, Incubation, Staining, Binding Assay, Concentration Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth *

doi: 10.1074/jbc.M112.389916

Figure Lengend Snippet: Coimmunoprecipitation of Olfm1 with NgR1 and LINGO-1. A and B, different forms of Olfm1 were cotransfected with FLAG-tagged NgR1, p75NTR, or LINGO-1. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody, and coprecipitated Olfm1 was detected in Western blot analysis (WB) with anti-Olfm1 antibody. All tested forms (AMZ, BMZ, and BMY) were coprecipitated with NgR1 (A). Olfm1 (AMZ) was also coprecipitated with LINGO-1 but not with p75NTR (B). All immunoprecipitation experiments were repeated at least three times. Input lines contained about 10% of lysates that were used for immunoprecipitation. C, validation of Olfm1 polyclonal antibody used for immunofluorescence study. Western blot analysis of lysates of COS7 cells transfected with the vector or the expression constructs encoding Olfm1, Olfm2, Olfm3, or different forms of Olfm1. The polyclonal Olfm1 antibody (1:2000) recognized only Olfm1 protein. The two right lanes show a Western blot analysis of mouse brain cortex (CX) or OB lysates. The antibody recognized one major band of Olfm1 protein in mouse CX and OB. The minor band represents Olfm1 dimers.

Article Snippet: The primary antibodies used were anti-Olfm1 (1:100 dilution), anti-NgR1 (1:50 dilution, Alomone Laboratory), and anti-EGFP (1:2,000 dilution, Aves Labs, Inc.).

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Transfection, Plasmid Preparation, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth *

doi: 10.1074/jbc.M112.389916

Figure Lengend Snippet: Colocalization of Olfm1 and NgR1 in mouse tissues and PC12 cells. A, DRG from E13.5 TG(Olfm1:EGFP) mice was stained with indicated antibodies. Upper panels, green, EGFP; red, NgR1. Lower panels, green, EGFP; red, Olfm1; purple, NgR1. Nuclei were stained with DAPI (blue). Note that only about 30% of NgR1-expressing cells were EGFP- and Olfm1-positive. Scale bar = 25 μm. B, retina from E13.5 (upper and center panels) and P3 (lower panels) TG(Olfm1:EGFP) mice stained for Olfm1 and NgR1. Colocalization of Olfm1 and NgR1 is observed in cell soma (arrows) and neurites (arrowheads). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bar = 25 μm. C, PC12 cells stained for Olfm1 (red) and NgR1 (green). Both Olfm1 and NgR1 accumulate in growth cones (arrowheads). Scale bars = 25 μm.

Article Snippet: The primary antibodies used were anti-Olfm1 (1:100 dilution), anti-NgR1 (1:50 dilution, Alomone Laboratory), and anti-EGFP (1:2,000 dilution, Aves Labs, Inc.).

Techniques: Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth *

doi: 10.1074/jbc.M112.389916

Figure Lengend Snippet: Inhibition of NgR1 complex formation by Olfm1. A, B, and C, Olfm1 and NgR1 ligands do not compete for binding with NgR1. A, Olfm1-AP (10 nm) binding to NgR1 with and without pretreatment with soluble MAG (40 nm) for 30 min. B, MAG-AP (10 nm) binding to NgR1 with or without pretreatment with Olfm1 (30 nm) for 30 min. C, quantification of Olfm1-AP (10 nm) binding to NgR1 after pretreatment with different NgR1 ligands. D and E, inhibition of Olfm1-AP binding to NgR1 by coexpression of p75NTR or LINGO-1. COS7 cells were transfected with NgR1 together with increasing amounts of p75NTR or LINGO-1. The expression levels of NgR1, p75NTR, and LINGO-1 in transfected cells were confirmed by Western blotting (lower panel in D). Olfm1-AP binding was quantified as described under “Experimental Procedures.” E, Scatchard plots of Olfm1 (AMZ)-AP binding to NgR1 in the presence of p75NTR or LINGO-1. These experiments were repeated three times, and results of typical experiment are shown. Blue circle, NgR1 alone; red square, NgR1 plus p75NTR; green triangle, NgR1 plus LINGO-1. F, inhibition of coimmunoprecipitation of NgR1 and p75NTR or LINGO-1 by Olfm1. COS7 cells were transfected with NgR1 together with indicated combinations of coreceptors. NgR1 was immunoprecipitated with anti-NgR1 antibody, and coprecipitated p75NTR or LINGO-1 were detected by Western blot analysis using corresponding antibodies.

Article Snippet: The primary antibodies used were anti-Olfm1 (1:100 dilution), anti-NgR1 (1:50 dilution, Alomone Laboratory), and anti-EGFP (1:2,000 dilution, Aves Labs, Inc.).

Techniques: Inhibition, Binding Assay, Transfection, Expressing, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth *

doi: 10.1074/jbc.M112.389916

Figure Lengend Snippet: Inhibition of NgR1-mediated and MAG-induced RhoA activation by Olfm1. A, COS7 cells were transfected with cDNAs encoding NgR1, p75 NTR and/or LINGO-1. 48 h after the transfection, purified Olfm1 or buffer control was added for 10 min, and then cells were stimulated with MAG-Fc (160 nm). Cells were harvested 15 min later, and the active form of RhoA (RhoA-GTP) was pulled down and detected by Western blotting. These experiments were repeated seven times. B, DRG growth cones were treated with MAG (80 nm) for 10 min with (lower panels) or without (center panels) Olfm1 (7 nm) pretreatment. Upper panels, untreated control. The explants were stained with anti-active RhoA antibody (red) together with phalloidin (green). Arrows show the direction of axon growth. Blue lines represent the outlines of growth cones. Scale bar = 5 μm. C, quantification of the fluorescence intensities for active RhoA in the immunostained growth cones (>30 growth cones from three to six explants) measured using ImageJ. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The primary antibodies used were anti-Olfm1 (1:100 dilution), anti-NgR1 (1:50 dilution, Alomone Laboratory), and anti-EGFP (1:2,000 dilution, Aves Labs, Inc.).

Techniques: Inhibition, Activation Assay, Transfection, Purification, Western Blot, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth *

doi: 10.1074/jbc.M112.389916

Figure Lengend Snippet: Inhibition of optic nerve growth on zebrafish embryos by olfm1 MO and rescues by ngr1 inhibition. A and B, zebrafish 1- to 4-cell-stage embryos were injected with or without indicated RNAs and olfm1 MO. As control RNA, EGFP RNA was injected. At 75 h post-fertilization, embryos were fixed, and DiI was injected to the retina in the unilateral eye. A, representative image of embryo head (frontal view) injected with DiI. The lower left round orange signals are the dye-injected position. Optic nerves are diagonally extended to the optic tectum (upper right panel) and make arborizations. B, optic nerve thickness was measured in the middle of the optic nerves and the percent arborizations were counted.

Article Snippet: The primary antibodies used were anti-Olfm1 (1:100 dilution), anti-NgR1 (1:50 dilution, Alomone Laboratory), and anti-EGFP (1:2,000 dilution, Aves Labs, Inc.).

Techniques: Inhibition, Injection

Journal: The Journal of Biological Chemistry

Article Title: Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth *

doi: 10.1074/jbc.M112.389916

Figure Lengend Snippet: Schematic diagrams illustrating the involvement of Olfm1 in the regulation of axon growth through the NgR1 complex and RhoA signaling. A, ligands such as MAG on the glial surface (bottom compartment) or unknown soluble molecules in the extracellular space (center compartment) bind to NgR1-p75NTR-LINGO-1 complex on the surface of the axonal growth cone (upper compartment). The signal is transduced to the intracellular space of the growth cone, converts RhoA to the active GTP-bound form, activates a cascade of proteins including ROCK and LIMK, and removes G-actin from cofilin. The released cofilin depolymerizes actin filaments, leading to growth cone collapse or retraction. B, when Olfm1 is secreted from the tips of the growth cone or neighboring cells, it binds to NgR1, dissociates NgR1-coreceptor interactions, and inhibits the activation of RhoA. This facilitates axon growth and may stimulate regeneration of damaged nerves.

Article Snippet: The primary antibodies used were anti-Olfm1 (1:100 dilution), anti-NgR1 (1:50 dilution, Alomone Laboratory), and anti-EGFP (1:2,000 dilution, Aves Labs, Inc.).

Techniques: Activation Assay

Journal: The Journal of Neuroscience

Article Title: Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination

doi: 10.1523/JNEUROSCI.1760-18.2019

Figure Lengend Snippet: Primary antibodies used in study

Article Snippet: Mouse anti-NgR 1:100 MAB1208 R&D Systems Human post-mortem MS brain immunofluorescence Rabbit anti-proteolipid (PLP) 1:500 Ab28486 Abcam Human post-mortem MS brain immunofluorescence Rabbit anti-pCRMP2-T555 1:40,000 ( Petratos et al., 2012 ; Mokhtar et al., 2018 ) Western blot Mouse anti-CRMP2 1:40,000 11096 IBL Western blot Rabbit anti-KIF5c 1:4000 LS- {"type":"entrez-nucleotide","attrs":{"text":"C81914","term_id":"33650884","term_text":"C81914"}} C81914 LSBio Western blot Mouse anti-β-actin 1:50,000 MAB1501 Millipore Western blot Mouse anti-Kinesin light chain-1 (KLC-1) 1:2000 MAB1617 Millipore Western blot Mouse anti-α-tubulin 1:10,000 05-829 Millipore Western blot Biotinylated anti-GFP 1:200 Ab6658 Abcam Oligodendrocyte apoptosis assay Mouse anti-CC1 1:50 {"type":"entrez-nucleotide","attrs":{"text":"M11217","term_id":"333925"}} M11217 Calbiochem Oligodendrocyte apoptosis assay Rabbit anti-cleaved caspase-3 1:400 9661 Cell Signaling Technology Oligodendrocyte apoptosis assay Open in a separate window Primary antibodies used in study table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Antibody Dilution Catalogue Supplier Application AlexaFluor 488 conjugated goat anti-rabbit IgG (H + L) 1:500 A-11008 Life Technologies Retina whole-mount, Optic nerve immunofluorescence AlexaFluor 546 conjugated goat anti-mouse IgG (H + L) 1:500 A-11030 Life Technologies Retina whole-mount, Optic nerve immunofluorescence, Oligodendrocyte apoptosis assay AlexaFluor 647 conjugated goat anti-rat IgG (H + L) 1:500 A-21247 Life Technologies Retina whole-mount, Optic nerve immunofluorescence, Oligodendrocyte apoptosis assay HRP-conjugated goat anti-mouse IgG (H + L) 1:20,000 AB308P Millipore Western blot HRP-conjugated goat anti-rabbit IgG (H + L) 1:20,000 AB307P Millipore Western blot Streptavidin, AlexaFluor 488 conjugated 1:500 {"type":"entrez-protein","attrs":{"text":"S11223","term_id":"112468","term_text":"pir||S11223"}} S11223 Life Technologies Oligodendrocyte apoptosis assay Open in a separate window Secondary antibodies used in study Quantitative PCR after reverse-transcription. mRNA was isolated from rAAV2-transduced retinae, and nontransduced retinae of naive ngr1 +/+ , ngr1 −/− , and ngr1 flx/flx mice 7 d post-intraocular injection by using RNeasy Mini kit (Qiagen) with DNase-I to remove genomic DNA.

Techniques: Immunofluorescence, Western Blot, Apoptosis Assay

Journal: Journal of Neurophysiology

Article Title: Differential expression of genes in the RhoA/ROCK pathway in the hippocampus and cortex following intermittent hypoxia and high-intensity interval training

doi: 10.1152/jn.00422.2023

Figure Lengend Snippet: TaqMan gene expression assay information (RhoA/ROCK pathway)

Article Snippet: Membrane receptors , NgR1 , Target , Rtn4r , Rn00586061_s1 , 57.

Techniques: Gene Expression, TaqMan Assay, Amplification, Membrane