Fig. 8. Analysis of Disp1 topology, localization and role in Shh secretion. (A) The predicted sequence of Disp1, with transmembrane helix highlighted in green and red. The blue-shaded sequence indicates what has been deleted in Disp1² mutation. (B) No signal was detected by immunostaining on unpermeabilized Cos7 cell using DspN and DspC antibodies. After permeabilization with Triton, Disp1 immunostaining were co-localized with ER marker Bip, and Golgi marker GM130. The protein also displayed a typical cell-surface distribution. (C) Proposed topology of Disp1 with sterol-sensing domain highlighted in red. (D) Immortalized Disp1^C829F/C829F mutant fibroblast was transfected with expression constructs as indicated (a). After 2 days, the media were collected and Shh protein was immuno-pulled down with anti-Shh 5E1 monoclonal antibody, separated by SDS-PAGE, and immuno-detected with anti-Shh AB80 polyclonal antibodies. (b) Cell-surface proteins were labeled by biotin and pulled down by streptavidin, then separated by SDS-PAGE and immuno-detected with AB80. L, cell lysate input before biotinylation; S, cell-surface fraction pulled down with streptavidin.