anti ngr1  (Alomone Labs)

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: Mouse Assay, 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: 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 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: 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