anced chemiluminescence reagent. The recombinant proteins were purified by affinity chromatography using a pre-charged Ni-NTA Sepharose column. The dialysis purity was verified by SDSPAGE, and the protein concentration was determined by the bicinchoninic acid protein quantitation method . Samples were stored at 220uC until use. Axon outgrowth and branch formation Glass coverslips were coated with poly-L-lysine, washed three times, and subsequently coated with NogoA FC or NogoA FC for 2 h at 37uC. Unbound NogoA was removed by three washes with PBS. To evaluate the blocking function of the mAbs, glass coverslips were coated with two mAbs for 1 h at 37uC. Primary hippocampal neurons cultures were prepared as previously described. Briefly, the hippocampal neurons were acquired from SD rat embryos. The pups were anesthetised, and 75% ethanol was sprayed on the animals for 5 min. The neurons were isolated and washed with D-Hank’s solution three times under sterile conditions. Cells were seeded at a density of 16106 cells/cm2 onto plates and maintained in a humidified incubator at 5% CO2 and 37uC. The neurons were cultured in Neurobasal supplemented with 2% B27, 1% glutamine, and 1% penicillin/streptomycin. Half of the medium was changed twice a week. The purity of neurons was determined by immunocytochemistry for bIII-tubulin, and the analysis indicated that 95% of the cells in the cultures were bIIItubulin positive. To observe axon outgrowth, cells were used for immunostaining on the seventh day after culture. The cells were washed after fixation in 4% PFA and then stained with anti-Map2 and 3 Antibodies of NogoA Enhance Axon Extension anti-Tau. Measurement of axon length was performed as follows. Five randomly chosen fields of view from the coverslips were photographed using a phase-contrast Olympus IMT2 microscope and an F-View camera at 206 magnification. The axon length per Tau-stained neuron was then measured from these photographs. Statistical significance was assessed using Student’s t test. The measurements of the total number of Taupositive neurons from three independent experiments were analysed by the t-test. To observe branch formation, the number of axon branch points of per neuron and the distance at which the axon sent out its first branches from the cell body were measured using a phasecontrast Olympus IMT2 microscope and an F-View camera at 406 after examining five randomly chosen fields of view from the coverslips. Statistical significance was assessed using Student’s t test. The measurements from three independent experiments were analysed by the t test. means 6 SEM. Differences between the groups were assessed by one-way ANOVA followed by the LSD-t test. P values,0.05 were considered to be significant. Results The two mAbs we MedChemExpress Piceatannol generated specifically recognised NogoA protein The specificity and the affinity of the two mAbs were tested by WB. The two mAbs and the commercial rabbit antiNogoA polyclonal antibody bound to NogoA from spinal cord tissue, with corresponding bands at 200 kDa. Additionally, aNogo66 mAb and aNogoA-N mAb, at different concentrations, strongly bound the NogoA molecule at 200 kDa, indicating that the two mAbs specifically recognise NogoA and have a good affinity for NogoA. NogoA localises to PubMed ID: the membrane surface, the cytoplasm, and processes in oligodendrocytes. First, IHC staining was used to determine the reactivity and specificity of the mAbs in spinal cord tissue from rats. The aNogo-N mAb and aNogo

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