Figure 4. Disp cleavage is required for Shh release.

(A) Disp-/- cells stably transfected with Shh or empty vector control were transiently transfected with GFP or the indicated DispHA expression vectors, and then co-cultured with LightII reporter cells. Reporter activity normalized to tk-renilla and relative to GFP control (set to 1) is shown. Assays were performed four times in duplicate or triplicate and all data points pooled. Error bars represent s.d. Significance was determined by one-way ANOVA. p***≤0.005. (B) Disp-/- mouse embryonic fibroblasts were stably transfected with empty vector control (V) or vector encoding Shh. Wild type or CS mutant Disp proteins were transiently expressed in Disp-/- cells alone or with Scube2, and lysates and media were examined by SDS-PAGE and western blot. Equal protein amounts (25 µg) from TCA precipitates of conditioned media were analyzed. Kinesin serves as loading control for lysate. Coomassie stain of membrane is shown as loading control for conditioned media (bottom). The graph (right) represents densitometry analysis of Shh media signal intensity normalized to media coomassie stain. The experiment was repeated three times. A representative experiment is shown. (C) Furin-/- cells were transiently transfected with Shh alone or with V5DispHA expression vector, and tested for Shh release as in (B). The experiment was repeated twice for Furin-/- Clone 2 and once for Clone 1, and a representative blot for Furin-/- Clone 2 is shown. The graph (right) represents densitometry analysis of Shh media signal normalized to media coomassie stain, and plotted relative to lysate Shh signal normalized to Actin. Analysis of the representative Clone 2 western is black. Densitometry analysis of the corresponding Clone 1 release assay is shown in gray.