Fig. 2. The N and C-terminal lipids act synergistically to restrict Shh to the membrane. (A) Anti-Shh western analysis of lysates derived from C17 cells expressing different lipid-containing Shh proteins. M, membrane bound (whole cell lysates); S, secreted (cell culture supernatants immunoprecipitated using 5E1 monoclonal antibody). (Lane 1) uShhN (7 ng; Shh made in E. coli containing no N- or C-terminal lipids); (lanes 2,3) C17 (non-transfected parental cell line); (lanes 4,5) C24S-Shh (C lipid alone); (lanes 6,7) wtShh (N+C lipid); (lanes 8,9) C24S-ShhN (no lipid); (lanes 10,11) ShhN (N lipid alone). Size standards are shown on the left. (B) The C17 neural cell lines expresses Ski1 and mouse dispatched, but not Ski2. cDNA isolated from C17 cells was subjected to PCR using primers against Ski1, Ski2 and dispatched (Disp), as indicated, and loaded on a 2% agarose gel stained with ethidium bromide to visualize PCR products.