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Fig. 1. Identification of N- and C-terminal fragments of human NgR expressed in SH-SY5Y cells. (A) SH-SY5Y cells expressing human NgR were incubated with OptiMEM for the indicated times and NgR in the lysate and medium detected by western blotting with the anti-V5 antibody. (B) Following SDS-PAGE, gel slices containing either the 64 (upper) or 48 (lower) kDa band of NgR in the cell lysate were excised and incubated with (+) or without (–) PNGase F followed by western blotting with the anti-V5 antibody. In parallel, medium from a 3 hour incubation with SH-SY5Y cells expressing NgR was treated with PNGase F and NgR was detected as above. (C) Cell lysate was incubated for 3 hours in the absence (–) or presence (+) of PI-PLC and treated with PNGase F followed by western blot analysis with the anti-V5 antibody. (D) Schematic of the human NgR construct used in the present study showing the N-terminal V5 tag (red) followed by the ligand-binding domain comprising the LRRNT (green), LRR (blue) and LRRCT (grey) sub-domains. Amino acid positions (italics) correspond to those for non-tagged human NgR. The epitope of the human NgR-specific monoclonal antibody (-hNgR-328) and the C-terminal GPI-anchor (GPI) are indicated. (E) Cell lysate and medium from a 3 hour incubation with SH-SY5Y cells expressing NgR and conditioned medium from a 72 hour incubation with HEK-293T cells expressing NgR-310 were subjected to western blot analysis with either the anti-V5 antibody, human NgR-specific monoclonal antibody (-hNgR-32B) or human NgR-specific polyclonal antibody (-hNgR poly).

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