Fig. 1

Active ShhN associates with the extracellular matrix. a Lysate and ECM deposited by mock and Shh-C199*-transfected Hek293t cells analyzed by SDS-PAGE/SYPRO-Ruby staining and on a Western Blot (H2 α-ShhN). Equal fractions of the total cell lysate and ECM were analyzed. ShhN is indicated. b Shh-C199*-transfected Hek293t cells were plated on glass slides and removed after 24 h. The slides were stained with mAb5E1, showing the presence of ShhN. Scale bar is 50 μm. c Supernatant and ECM conditioned by Shh-C199*-transfected Hek293t cells. LightII cells were either grown on mock or ShhN conditioned ECM. Cells grown on ECM deposited by mock transfected cell were grown in the absence or presence of mock or Shh-C199* conditioned supernatant. Box and whisker plots, n ≥ 3. *p < 0.05, ****p < 0.0001. d Hh response in LightII cells grown on the decellularized ECM of mock-, Shh-C199*-, or Shh-transfected Hek293t cells. ****p < 0.0001. e Western blot analysis of Hek293t cells transfected with the indicated Shh mutants. 100 nM MG-132 (proteasome inhibitor), 100 nM Chloroquine and 100 nM Concanamycin A (inhibitors of endosome acidification) were assessed for their ability to affect Shh accumulation. Immune reactive signals are quantified and normalized to the untreated and Shh-C199* transfected condition. f Western blot analysis of Hek293t cells transfected with the indicated Shh mutants, and the effects of the dynamin inhibitor Dynasore (50 μM) was assessed for its effect on Shh accumulation. Immune reactive signals are quantified and normalized to the untreated and Shh-C199* transfected condition. Full-length gels blots are presented in Supplementary Figure 3