Figure 1. Shh is secreted in lipoprotein-associated and lipoprotein-free forms.

(A) Density of human Shh secreted by HeLa cells in the absence or presence of fetal bovine serum (FBS), analyzed by Optiprep density gradient centrifugation, and Western blotting (WB). HeLa cells transfected with Shh were grown in serum-free medium or in the presence of 10% FBS, and equal volumes of supernatants analyzed. Colors indicate fractions corresponding to bovine Very Low-, Low-, and High-Density Lipoproteins (VLDL, LDL, and HDL) [68]. (B) Density of non-lipid-modified Shh-N^C24S, analyzed by Optiprep density gradient centrifugation and WB. Supernatants were derived from HeLa cells transfected with Shh-N^C24S and grown in the presence of FBS. (C) Shh levels in cell lysates and supernatants derived from HeLa cells transfected with Shh, grown in serum-free medium supplemented with individual human lipoprotein classes. Equal protein amounts (cell lysates) or volumes (supernatants) were analyzed. (D) Density of Shh in HeLa cell supernatants shown in (C), analyzed by Optiprep density gradient centrifugation and WB. Colors indicate fractions corresponding to human VLDL, LDL, and HDL [43]. (E) Co-Immunoprecipitation (Co-IP) of secreted Shh with different lipoprotein classes, analyzed by WB. Supernatants were derived from HeLa cells transfected with Shh or Shh-N^C24S, grown in serum-free medium supplemented with individual human lipoproteins classes. (F) Shh levels in supernatants derived from MIA PaCa-2 cells grown in serum-free medium supplemented with individual human lipoprotein classes. Equal volumes were used for WB. (G) Density of Shh in MIA PaCa-2 cell supernatants shown in (F), analyzed by Optiprep density gradient centrifugation and WB. (H) Density of Shh in supernatants from Shh-expressing HeLa cells grown in serum-free medium supplemented with hemolymph from Drosophila larvae, analyzed by Optiprep density gradient centrifugation and WB. Purple indicates fractions corresponding to Drosophila Lpp.