Fig. 4.

HH fusion constructs demonstrate the HH C-terminus defines auto-processing and secretion events. (A) Schematic representation of fusion cDNA constructs. (B) Luciferase assays were performed on Light2 cells treated for 48 h with conditioned media from 293T cells transfected with full length SHH and HH fusion constructs (1:4 in media with 0.5% FBS). Luciferase activity is shown as a % of activity for full length SHH. (C) Gli1 protein levels were determined by Western blot of cell lysates from 3T3 cells treated with conditioned media from 293T cells transfected with HH and HH fusion constructs (1:4 in media with 0.5% FBS). (D) The presence of secreted HH was determined Western blot of conditioned media from transfected 293T cells cultured for 48 h in media with 2% FBS. (E) HH proteins were detected by Western blot using the 5E1 anti-HH antibody on cell lysates from transfected 293T cells cultured for 48 h in media with 2% FBS. (F) Luciferase assays were performed on Light2 cells co-plated for 48 h in media with 0.5% FBS with transfected 293T cells. Luciferase activity is shown as a % of activity for 293T cells transfected with full length SHH.