Genotypes from the offspring from the mutant mice were assessed with PCR using the next primer sequences to focus on the intronic area from the mouse lotus locus (forwards: 5-Label CTC TTC TCC CGG GAA GC-3, change: 5-CTT GCA CCC ATC CCA GAA GG-3) or the genomic area containing the neomycin gene (forwards: 5-GGA TTC ATC GAC TGT GGC CG-3). Through the entire experimental procedures, all efforts were designed to minimize the amount of animals used and their suffering. analyses uncovered that s-LOTUS inhibited MAI-induced development cone collapse and neurite outgrowth inhibition in chick DRG neurons. Furthermore, whereas olfactory light bulb neurons of mutant mice had been generated as defined previously (Sato et al., 2011) and had been housed within a specific-pathogen-free service with usage of autoclaved food and water. Genotypes from the offspring from the mutant mice had been evaluated with PCR using the next primer sequences to focus on the intronic area from the mouse lotus locus (forwards: 5-Label CTC TTC TCC CGG GAA GC-3, invert: 5-CTT GCA CCC ATC CCA GAA GG-3) or the genomic area filled with the neomycin gene (forwards: 5-GGA TTC ATC GAC TGT GGC CG-3). Through the entire experimental techniques, all efforts had been designed to minimize the amount of pets utilized and their struggling. The procedures had been accepted by the institutional pet care and make use of ethics committee of Yokohama Town University (acceptance amount: T13-001, T13-009, T-A-15-001, T-A-16-002). Structure of appearance plasmids. The plasmids encoding 6His normally label- and alkaline phosphatase (AP) tag-fused Nogo66 (6His-AP-Nogo66), which can be an NgR1-binding domains and an axonal development inhibitory domains of Nogo-A (GrandPr et al., 2000) or the extracellular domains of mouse MAG fusing AP label and 6His normally label (MAG-AP-6His) had been generated as defined previously (Sato et al., 2011; Kurihara et al., 2014). The plasmid encoding 6His normally label- and AP tag-fused glycosylphosphatidylinositol (GPI)-anchored site-deleted individual OMgp (AP-OMgp) was supplied by Stephen M. Strittmatter AM-2099 (Barton et al., 2003; Li et al., 2004). The plasmids encoding full-length mouse LOTUS or mouse NgR1 had been generated as defined previously (Sato et al., 2011). The mouse p75NTR cDNA was amplified by PCR from a mouse adult human brain cDNA template and cloned right into a mammalian appearance vector. The transmembrane region-deleted mouse LOTUS fusing AM-2099 mutated individual Fc label constructed to lessen antibody-dependent mobile cytotoxicity and complement-dependent cytotoxicity accompanied by 6His normally label (LOTUS-Fc-6His), streptavidin-binding peptide (SBP) label- as well as the above individual Fc tag-fused mouse LOTUS without its transmembrane area (SBP-Fc-LOTUS), SBP label- and FLAG tag-fused mouse LOTUS (SBP-FLAG-LOTUS), GPI-linkage region-deleted mouse NgR1 (Fournier et al., 2001) fusing the above mentioned individual Fc label and SBP label (NgR1-Fc-SBP), the SBP label- as well as the over individual Fc tag-fused ectodomain of mouse p75NTR (SBP-Fc-p75NTR), GST tag-fused p75NTR (GST-p75NTR), as well as the SBP label- and AP tag-fused ectodomain of mouse MAG (SBP-AP-MAG) had been constructed by regular PCR cloning using the KOD DNA polymerase (TOYOBO). All fragments placed in each plasmid had been verified by DNA sequencing. The accession amounts of these mRNAs are referred to as follows: mouse LOTUS (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_145123″,”term_id”:”1253558211″,”term_text”:”NM_145123″NM_145123), mouse p75NTR (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_033217″,”term_id”:”118131161″,”term_text”:”NM_033217″NM_033217), mouse NgR1 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_022982″,”term_id”:”1379048533″,”term_text”:”NM_022982″NM_022982), mouse Nogo-A (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_194054″,”term_id”:”281306800″,”term_text”:”NM_194054″NM_194054), mouse MAG (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_010758″,”term_id”:”1070257828″,”term_text”:”NM_010758″NM_010758), and human OMgp (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002544″,”term_id”:”1519245611″,”term_text”:”NM_002544″NM_002544). Cell culture. Cos-7 cells (RRID:CVCL_0024) and HEK293T (RRID:CVCL_0063) cells were cultured in DMEM (4.5 g/L glucose with l-glutamine and sodium pyruvate; Nacalai Tesque) made up of 10% FBS (Biological Industries) and 0.5% penicillinCstreptomycin mixed solution (Nacalai Tesque). For cell culture, all of the cells were manipulated with sterile technique and incubated at 37C with 5% CO2. Protein purification. The plasmids (0.35 g/ml) encoding the SBP tag or 6His tag sequences were transfected with polyethylenimine Max (2 g/ml; Polysciences) into HEK293T cells and cells were incubated for 4 d. Cell culture medium was centrifuged at 117,000 for 1 h. SBP-tagged protein was collected from the culture medium using high capacity streptavidin agarose resin (Thermo Fisher Scientific, catalog #20361) and eluted with PBS made up of 2 mm biotin. The centrifuged supernatant of the culture medium made up of 6His-tagged protein was dialyzed in 20 mm Tris, pH 7.6, and 500 mm NaCl for 3 h twice. 6His-tagged protein was adsorbed to Talon metal affinity resin (Clontech, catalog #635502) and eluted with the above dialysis answer made up of 50 mm imidazole. AP-fused protein was added to for 10 min, the supernatant was incubated with glutathione Sepharose beads (10 l per sample; GE Healthcare, catalog #17-0756-01) and incubated for 4 h at 4C with moderate rotation. For s-LOTUS treatment, the transfected cells were treated with SBP-Fc-LOTUS (1 m) or its vehicle (control) diluted with culture medium and incubated for 30 min at 37C with 5% CO2. After cell lysate was prepared, SBP-Fc-LOTUS or its vehicle was applied to the cell lysate at a concentration of 1 1 m with glutathione Sepharose beads and the mixture was incubated for 4 h at.Masumi Iketani at Tokyo Metropolitan Institute of Gerontology for skillful technical assistance with the primary culture of OB neurons, Ms. food. Genotypes of the offspring of the mutant mice were assessed with PCR using the following primer sequences to target the intronic region of the mouse lotus locus (forward: 5-TAG CTC TTC TCC CGG GAA GC-3, reverse: 5-CTT GCA CCC ATC CCA GAA GG-3) or the genomic region made up of the neomycin gene (forward: 5-GGA TTC ATC GAC TGT GGC CG-3). Throughout the experimental procedures, all efforts were made to minimize the number of animals used and their suffering. The procedures were approved by the institutional animal care and use ethics committee of Yokohama City University (approval number: T13-001, T13-009, T-A-15-001, T-A-16-002). Construction of expression plasmids. The plasmids encoding 6His usually tag- and alkaline phosphatase (AP) tag-fused Nogo66 (6His-AP-Nogo66), which is an NgR1-binding domain name and an axonal growth inhibitory domain name of Nogo-A (GrandPr et al., 2000) or the extracellular domain name of mouse MAG fusing AP tag and 6His usually tag (MAG-AP-6His) were generated as described previously (Sato et al., 2011; Kurihara et al., 2014). The plasmid encoding 6His usually tag- and AP tag-fused glycosylphosphatidylinositol (GPI)-anchored site-deleted human OMgp (AP-OMgp) was provided by Stephen M. Strittmatter (Barton et al., 2003; Li et al., 2004). The plasmids encoding full-length mouse LOTUS or mouse NgR1 were generated as described previously (Sato et al., 2011). The mouse p75NTR cDNA was amplified by PCR from a mouse adult brain cDNA template and cloned into a mammalian expression vector. The transmembrane region-deleted mouse LOTUS fusing mutated human Fc tag constructed to reduce antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity followed by 6His usually tag (LOTUS-Fc-6His), streptavidin-binding peptide (SBP) tag- and the above human Fc tag-fused mouse LOTUS without its transmembrane region (SBP-Fc-LOTUS), SBP tag- and FLAG tag-fused mouse LOTUS (SBP-FLAG-LOTUS), GPI-linkage region-deleted mouse NgR1 (Fournier et al., 2001) fusing the above human Fc tag and SBP tag (NgR1-Fc-SBP), the SBP tag- and the above human Fc tag-fused ectodomain of mouse p75NTR (SBP-Fc-p75NTR), GST tag-fused p75NTR (GST-p75NTR), and the SBP tag- and AP tag-fused ectodomain of mouse MAG (SBP-AP-MAG) were constructed by standard PCR cloning using the KOD DNA polymerase (TOYOBO). All fragments inserted in each plasmid were confirmed by DNA sequencing. The accession numbers of these mRNAs are described as follows: mouse LOTUS (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_145123″,”term_id”:”1253558211″,”term_text”:”NM_145123″NM_145123), mouse p75NTR (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_033217″,”term_id”:”118131161″,”term_text”:”NM_033217″NM_033217), mouse NgR1 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_022982″,”term_id”:”1379048533″,”term_text”:”NM_022982″NM_022982), mouse Nogo-A (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_194054″,”term_id”:”281306800″,”term_text”:”NM_194054″NM_194054), mouse MAG (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_010758″,”term_id”:”1070257828″,”term_text”:”NM_010758″NM_010758), and human OMgp (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002544″,”term_id”:”1519245611″,”term_text”:”NM_002544″NM_002544). Cell culture. Cos-7 cells (RRID:CVCL_0024) and HEK293T (RRID:CVCL_0063) cells were cultured in DMEM (4.5 g/L glucose with l-glutamine and sodium pyruvate; Nacalai Tesque) containing 10% FBS (Biological Industries) and 0.5% penicillinCstreptomycin mixed solution (Nacalai Tesque). For cell culture, all of the cells were manipulated with sterile technique and incubated at 37C with 5% CO2. Protein purification. The plasmids (0.35 g/ml) encoding the SBP tag or 6His tag sequences were transfected with polyethylenimine Max (2 g/ml; Polysciences) into HEK293T cells and cells were incubated for 4 d. Cell culture medium was centrifuged at 117,000 for 1 h. SBP-tagged protein was collected from the culture medium using high capacity streptavidin agarose resin (Thermo Fisher Scientific, catalog #20361) and eluted with PBS containing 2 mm biotin. The centrifuged supernatant of the culture medium containing 6His-tagged protein was dialyzed in 20 mm Tris, pH 7.6, and 500 mm NaCl for 3 h twice. 6His-tagged protein was adsorbed to Talon metal affinity resin (Clontech, catalog #635502) and eluted with the above dialysis solution containing 50 mm imidazole. AP-fused protein was added to for 10 min, the supernatant was incubated with glutathione Sepharose beads (10 l per sample; GE Healthcare, catalog #17-0756-01) and incubated for 4 h at 4C with mild rotation. For s-LOTUS treatment, the transfected cells were treated with SBP-Fc-LOTUS (1 m) or its vehicle (control) diluted with culture medium and incubated for 30 min at 37C with 5% CO2. After cell lysate was prepared, SBP-Fc-LOTUS or its vehicle was applied to the cell lysate at a concentration of 1 1 m with glutathione Sepharose beads and the mixture was incubated for 4 h at 4C. The beads were washed with the lysis buffer four times, suspended with 4 Laemmli buffer, and boiled for 7 min at 100C. RhoA pull-down assay. Cortical neurons of embryonic day.Cos-7 cells (RRID:CVCL_0024) and HEK293T (RRID:CVCL_0063) cells were cultured in DMEM (4.5 g/L glucose with l-glutamine and sodium pyruvate; Nacalai Tesque) containing 10% FBS (Biological Industries) and 0.5% penicillinCstreptomycin mixed solution (Nacalai Tesque). NgR1. s-LOTUS inhibited myelin-associated inhibitor (MAI)-induced RhoA activation in murine cortical neurons. Functional analyses revealed that s-LOTUS inhibited MAI-induced growth cone collapse and neurite outgrowth inhibition in chick DRG neurons. In addition, whereas olfactory bulb neurons of mutant mice were generated as described previously (Sato et al., 2011) and were housed in a specific-pathogen-free facility with access to autoclaved water and food. Genotypes of the offspring of the mutant mice were assessed with PCR using the following primer sequences to target the intronic region of the mouse lotus locus (forward: 5-TAG CTC TTC TCC CGG GAA GC-3, reverse: 5-CTT GCA CCC ATC CCA GAA GG-3) or the genomic region containing the neomycin gene (forward: 5-GGA TTC ATC GAC TGT GGC CG-3). Throughout the experimental procedures, all efforts were made to minimize the number of animals used and their suffering. The procedures were approved by the institutional animal care and use ethics committee of Yokohama City University (approval number: T13-001, T13-009, T-A-15-001, T-A-16-002). Construction of expression plasmids. The plasmids encoding 6His tag- and alkaline phosphatase (AP) tag-fused Nogo66 (6His-AP-Nogo66), which is an NgR1-binding domain and an axonal growth inhibitory domain of Nogo-A (GrandPr et al., 2000) or the extracellular domain of mouse MAG fusing AP tag and 6His tag (MAG-AP-6His) were generated as described previously (Sato et al., 2011; Kurihara et al., 2014). The plasmid encoding 6His tag- and AP tag-fused glycosylphosphatidylinositol (GPI)-anchored site-deleted human OMgp (AP-OMgp) was provided by Stephen M. Strittmatter (Barton et al., 2003; Li et al., 2004). The plasmids encoding full-length mouse LOTUS or mouse NgR1 were generated as described previously (Sato et al., 2011). The mouse p75NTR cDNA was amplified by PCR from a mouse adult brain cDNA template and cloned into a mammalian expression vector. The transmembrane region-deleted mouse LOTUS fusing mutated human Fc tag constructed to reduce antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity followed by 6His tag (LOTUS-Fc-6His), streptavidin-binding peptide (SBP) tag- and the above human Fc tag-fused mouse LOTUS without its transmembrane region (SBP-Fc-LOTUS), SBP tag- and FLAG tag-fused mouse LOTUS (SBP-FLAG-LOTUS), GPI-linkage region-deleted mouse NgR1 (Fournier et P21 al., 2001) fusing the above human being Fc tag and SBP tag (NgR1-Fc-SBP), the SBP tag- and the above human being Fc tag-fused ectodomain of mouse p75NTR (SBP-Fc-p75NTR), GST tag-fused p75NTR (GST-p75NTR), and the SBP tag- and AP tag-fused ectodomain of mouse MAG (SBP-AP-MAG) were constructed by standard PCR cloning using the KOD DNA polymerase (TOYOBO). All fragments put in each plasmid were confirmed by DNA sequencing. The accession numbers of these mRNAs are described as follows: mouse LOTUS (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_145123″,”term_id”:”1253558211″,”term_text”:”NM_145123″NM_145123), mouse p75NTR (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_033217″,”term_id”:”118131161″,”term_text”:”NM_033217″NM_033217), mouse NgR1 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_022982″,”term_id”:”1379048533″,”term_text”:”NM_022982″NM_022982), mouse Nogo-A (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_194054″,”term_id”:”281306800″,”term_text”:”NM_194054″NM_194054), mouse MAG (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_010758″,”term_id”:”1070257828″,”term_text”:”NM_010758″NM_010758), and human being OMgp (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002544″,”term_id”:”1519245611″,”term_text”:”NM_002544″NM_002544). Cell tradition. Cos-7 cells (RRID:CVCL_0024) and HEK293T (RRID:CVCL_0063) cells were cultured in DMEM (4.5 g/L glucose with l-glutamine and sodium pyruvate; Nacalai Tesque) comprising 10% FBS (Biological Industries) and 0.5% penicillinCstreptomycin mixed solution (Nacalai Tesque). For cell tradition, all the cells were manipulated with sterile technique and incubated at 37C with 5% CO2. Protein purification. The plasmids (0.35 g/ml) encoding the SBP tag or 6His tag sequences were transfected with polyethylenimine Max (2 g/ml; Polysciences) into HEK293T cells and cells were incubated for 4 d. Cell tradition medium was centrifuged at 117,000 for 1 h. SBP-tagged protein was collected from your tradition medium using high capacity streptavidin agarose resin (Thermo Fisher Scientific, catalog #20361) and eluted with PBS comprising 2 mm biotin. The centrifuged supernatant of the tradition medium comprising 6His-tagged protein was dialyzed in 20 mm Tris, pH 7.6, and 500 mm NaCl for 3 h twice. 6His-tagged protein was adsorbed to Talon metallic affinity resin (Clontech, catalog #635502) and eluted with the above dialysis remedy comprising 50 mm imidazole. AP-fused protein was added to for 10 min, the supernatant was incubated with glutathione Sepharose beads (10 l per sample; GE Healthcare, catalog #17-0756-01) and incubated for 4 h at 4C with slight rotation. For s-LOTUS treatment, the transfected cells were treated with SBP-Fc-LOTUS (1 m) or its vehicle (control) diluted with tradition medium and incubated for 30 min at 37C with 5% CO2. After cell lysate was prepared,.Therefore, LOTUS is definitely expected to have therapeutic potential for the promotion of neuronal regeneration. explained previously (Sato et al., 2011) and were housed inside a specific-pathogen-free facility with access to autoclaved water and food. Genotypes of the offspring of the mutant mice were assessed with PCR using the following primer sequences to target the intronic region of the mouse lotus locus (ahead: 5-TAG CTC TTC TCC CGG GAA GC-3, reverse: 5-CTT GCA CCC ATC CCA GAA GG-3) or the genomic region comprising the neomycin gene (ahead: 5-GGA TTC ATC GAC TGT GGC CG-3). Throughout the experimental methods, all efforts were made to minimize the number of animals used and their suffering. The procedures were authorized by the institutional animal care and use ethics committee of Yokohama City University (authorization quantity: T13-001, T13-009, T-A-15-001, T-A-16-002). Building of manifestation plasmids. The plasmids encoding 6His definitely tag- and alkaline phosphatase (AP) tag-fused Nogo66 (6His-AP-Nogo66), which is an NgR1-binding website and an axonal growth inhibitory website of Nogo-A (GrandPr et al., 2000) or the extracellular website of mouse MAG fusing AP tag and 6His definitely tag (MAG-AP-6His) were generated as explained previously (Sato et al., 2011; Kurihara et al., 2014). The plasmid encoding 6His definitely tag- and AP tag-fused glycosylphosphatidylinositol (GPI)-anchored site-deleted human being OMgp (AP-OMgp) was provided by Stephen M. Strittmatter (Barton et al., 2003; Li et al., 2004). The plasmids encoding full-length mouse LOTUS or mouse NgR1 were generated as explained previously (Sato et al., 2011). The mouse p75NTR cDNA was amplified by PCR from a mouse adult mind cDNA template and cloned into a mammalian manifestation vector. The transmembrane region-deleted mouse LOTUS fusing mutated human being Fc tag constructed to reduce antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity followed by 6His definitely tag (LOTUS-Fc-6His), streptavidin-binding peptide (SBP) tag- and the above human being Fc tag-fused mouse LOTUS without its transmembrane region (SBP-Fc-LOTUS), SBP tag- and FLAG tag-fused mouse LOTUS (SBP-FLAG-LOTUS), GPI-linkage region-deleted mouse NgR1 (Fournier et al., 2001) fusing the above human being Fc tag and SBP tag (NgR1-Fc-SBP), the SBP tag- and the above human Fc tag-fused ectodomain of mouse p75NTR (SBP-Fc-p75NTR), GST tag-fused p75NTR (GST-p75NTR), and the SBP tag- and AP tag-fused ectodomain of mouse MAG (SBP-AP-MAG) were constructed by standard PCR cloning using the KOD DNA polymerase (TOYOBO). All fragments inserted in each plasmid were confirmed by DNA sequencing. The accession numbers of these mRNAs are described as follows: mouse LOTUS (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_145123″,”term_id”:”1253558211″,”term_text”:”NM_145123″NM_145123), mouse p75NTR (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_033217″,”term_id”:”118131161″,”term_text”:”NM_033217″NM_033217), mouse NgR1 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_022982″,”term_id”:”1379048533″,”term_text”:”NM_022982″NM_022982), mouse Nogo-A (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_194054″,”term_id”:”281306800″,”term_text”:”NM_194054″NM_194054), mouse MAG (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_010758″,”term_id”:”1070257828″,”term_text”:”NM_010758″NM_010758), and human OMgp (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002544″,”term_id”:”1519245611″,”term_text”:”NM_002544″NM_002544). Cell culture. Cos-7 cells (RRID:CVCL_0024) and HEK293T (RRID:CVCL_0063) cells were cultured in DMEM (4.5 g/L glucose with l-glutamine and sodium pyruvate; Nacalai Tesque) made up of 10% FBS (Biological Industries) and 0.5% penicillinCstreptomycin mixed solution (Nacalai Tesque). For cell culture, all of the cells were manipulated with sterile technique and incubated at 37C with 5% CO2. Protein purification. The plasmids (0.35 g/ml) encoding the SBP tag or 6His tag sequences were transfected with polyethylenimine Max (2 g/ml; Polysciences) into HEK293T cells and cells were incubated for 4 d. Cell culture medium was centrifuged at 117,000 for 1 h. SBP-tagged protein was collected from your culture medium using high capacity streptavidin agarose resin (Thermo Fisher Scientific, catalog #20361) and eluted with PBS made up of 2 mm biotin. The centrifuged supernatant of the culture medium made up of 6His-tagged protein was dialyzed in 20 mm Tris, pH 7.6,.** 0.01 versus control; two-way ANOVA followed by Tukey analysis. Discussion Our present study showed that s-LOTUS binds to both p75NTR and NgR1 and interferes with the molecular interaction between p75NTR and NgR1, resulting in the suppression of MAI-induced RhoA activation, growth cone collapse, and neurite outgrowth inhibition and promotes axonal regeneration after optic nerve injury em in vivo /em . p75NTR and NgR1. s-LOTUS inhibited myelin-associated inhibitor (MAI)-induced RhoA activation in murine cortical neurons. Functional analyses revealed that s-LOTUS inhibited MAI-induced growth cone collapse and neurite outgrowth inhibition in chick DRG neurons. In addition, whereas olfactory bulb neurons of mutant mice were generated as explained previously (Sato et al., 2011) and were housed in a specific-pathogen-free facility with access to autoclaved water and food. Genotypes of the offspring of the mutant mice were assessed with PCR using the following primer sequences to target the intronic region of the mouse lotus locus (forward: 5-TAG CTC TTC TCC CGG GAA GC-3, reverse: 5-CTT GCA CCC ATC CCA GAA GG-3) or the genomic region made up of the neomycin gene (forward: 5-GGA TTC ATC GAC TGT GGC CG-3). Throughout the experimental procedures, all efforts were made to minimize the number of animals used and their suffering. The procedures were approved by the institutional animal care and use AM-2099 ethics committee of Yokohama City University (approval number: T13-001, T13-009, T-A-15-001, T-A-16-002). Construction of expression plasmids. The plasmids encoding 6His usually tag- and alkaline phosphatase (AP) tag-fused Nogo66 (6His-AP-Nogo66), which is an NgR1-binding domain name and an axonal growth inhibitory domain name of Nogo-A (GrandPr et al., 2000) or the extracellular domain name of mouse MAG fusing AP tag and 6His usually tag (MAG-AP-6His) were generated as explained previously (Sato et al., 2011; Kurihara et al., 2014). The plasmid encoding 6His usually tag- and AP tag-fused glycosylphosphatidylinositol (GPI)-anchored site-deleted human OMgp (AP-OMgp) was provided by Stephen M. Strittmatter (Barton et al., 2003; Li et al., 2004). The plasmids encoding full-length mouse LOTUS or mouse NgR1 were generated as explained previously (Sato et al., 2011). The mouse p75NTR cDNA was amplified by PCR from a mouse adult brain cDNA template and cloned into a mammalian expression vector. The transmembrane region-deleted mouse LOTUS fusing mutated human Fc tag constructed to reduce antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity followed by 6His usually tag (LOTUS-Fc-6His), streptavidin-binding peptide (SBP) tag- and the above human Fc tag-fused mouse LOTUS without its transmembrane region (SBP-Fc-LOTUS), SBP tag- and FLAG tag-fused mouse LOTUS (SBP-FLAG-LOTUS), GPI-linkage region-deleted mouse NgR1 (Fournier et al., 2001) fusing the above human Fc tag and SBP tag (NgR1-Fc-SBP), the SBP tag- and the above human Fc tag-fused ectodomain of mouse p75NTR (SBP-Fc-p75NTR), GST tag-fused p75NTR (GST-p75NTR), and the SBP tag- and AP tag-fused ectodomain of mouse MAG (SBP-AP-MAG) were constructed by standard PCR cloning using the KOD DNA polymerase (TOYOBO). All fragments inserted in each plasmid were confirmed by DNA sequencing. The accession numbers of these mRNAs are described as follows: mouse LOTUS (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_145123″,”term_id”:”1253558211″,”term_text”:”NM_145123″NM_145123), mouse p75NTR (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_033217″,”term_id”:”118131161″,”term_text”:”NM_033217″NM_033217), mouse NgR1 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_022982″,”term_id”:”1379048533″,”term_text”:”NM_022982″NM_022982), mouse Nogo-A (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_194054″,”term_id”:”281306800″,”term_text”:”NM_194054″NM_194054), mouse MAG (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_010758″,”term_id”:”1070257828″,”term_text”:”NM_010758″NM_010758), and human OMgp (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002544″,”term_id”:”1519245611″,”term_text”:”NM_002544″NM_002544). Cell culture. Cos-7 cells (RRID:CVCL_0024) and HEK293T (RRID:CVCL_0063) cells were cultured in DMEM (4.5 g/L glucose with l-glutamine and sodium pyruvate; Nacalai Tesque) made up of 10% FBS (Biological Industries) and 0.5% penicillinCstreptomycin mixed solution (Nacalai Tesque). For cell culture, all of the cells were manipulated with sterile technique and incubated at 37C with 5% CO2. Protein purification. The plasmids (0.35 g/ml) encoding the SBP label or 6His label sequences were transfected with polyethylenimine Max (2 g/ml; Polysciences) into HEK293T cells and cells had been incubated for 4 d. Cell tradition moderate was centrifuged at 117,000 for 1 h. SBP-tagged proteins was collected through the culture moderate using high capability streptavidin agarose resin (Thermo Fisher Scientific, catalog #20361) and eluted with PBS including 2 mm biotin. The centrifuged supernatant from the culture medium.