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Fig. 2. CTF-NgR is at the cell surface and associated with lipid rafts. (A) SH-SY5Y cells expressing NgR were surface biotinylated and biotinylated proteins precipitated from the cell lysate with streptavidin-agarose. NgR and clathrin were detected in the input lysate (LYS) and streptavidin-agarose precipitate (STREP PPT) using the human NgR-specific polyclonal antibody and anti-clathrin antibody, respectively. (B) Cells were homogenised in the presence of 1% Triton-100 and subjected to buoyant sucrose density centrifugation to isolate lipid rafts. Following centrifugation, the gradient was separated into eight fractions, with fraction 1 containing the highest density of sucrose. Equivalent volumes from each fraction were subjected to SDS-PAGE and western blot analysis with the indicated antibodies. NgR was detected by either the anti-V5 antibody or the human NgR-specific polyclonal antibody.





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