FIGURE 7.

C-terminal tags impair ShhNC autocatalytic cholesteroylation and regulated Shh release. (A) Representative Western blots of C-terminally modified Shh variants. Solubilized Shh (supernatant) and cellular Shh (cell lysates) were blotted and stained with antibodies directed against Shh or its C-terminal tags. (B) Quantification of autocatalytic cholesteroylation efficiencies. Ratios of processed cellular Shh relative to precursors were determined and processing of the untagged protein was set to 1 (black bar). (C) Quantification of Shh release. Ratios of soluble (supernatant) Shh relative to cellular (cell lysate) Shh were determined and untagged protein release was set to 1 (black bar). Mean values ± SD are shown. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple-comparison test. ns: not significant *P < 0.05 ***P < 0.001. (D) Bioactivity of Shh and its C-terminally modified variants. Non-cholesterol modified but C-terminally tagged Shh was expressed in Bosc23 cells and subsequently induced osteoblast differentiation of C3H10 T1/2 reporter cells. C-terminal protein tags did not interfere with Ptc receptor binding. (E,G) Representative Western blots of Shh or its variants with or without Scube2 and inactive Scube2^ΔCUB. (F) Quantification of (E,G). Mean values ± SD are shown in all graphs. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple-comparison test. ns: not significant, *P < 0.05, ***P < 0.001.