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Fig. 6. NTF-NgR binds Nogo-66 but not p75^NTR and inhibits Nogo-66 binding to cells expressing NgR. (A) Medium from a 3 hour incubation with equivalent amounts of mock transfected SH-SY5Y or SH-SY5Y cells expressing NgR was subjected to immunoprecipitation with the anti-V5 antibody and equivalent amounts of precipitate incubated at room temperature for 2 hours in M-PER containing 10 nM AP (white bar) or AP-Nogo-66 (black bars). Following four washes in TNS buffer, the amount of AP activity associated with the precipitate was quantified using 1-Step^TM PNPP. The results are the mean±s.d. of three independent experiments. (B) 1 nM AP or AP-Nogo-66 were pre-incubated with medium from a 3 hour incubation with mock transfected SH-SY5Y (black bars) or SH-SY5Y expressing NgR (white bars) and subsequently incubated with CHO cells stably expressing human NgR. Cells were washed, fixed and the amount of AP activity associated with the cells quantified as above. The results are the mean±s.d. of three independent experiments. (C) Lysate from SH-SY5Y cells expressing Ng R was subjected to immunoprecipitation (IP) with either the anti-p75^NTR antibody or an unrelated IgG antibody. NgR and p75^NTR were detected in the input lysate and immunoprecipitate using the anti-V5 and anti-p75^NTR antibodies, respectively.

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