Figure 5

The effects of Hh protein and agonist on vertebrate Smo and Ptc proteins. A stable cell line expressing Ptc-GFP and HA-Smo retroviral constructs was generated to evaluate the effects of Hh protein versus agonist on the Hh receptor components. (a) Anti-Ptc protein blot of anti-GFP immunoprecipitates, fractionated by SDS-polyacrylamide gel electrophoresis, from cells treated with vehicle (lanes 1,4,7), 25 nM Hh protein (lanes 2,5,8) or 0.2 μM Hh-Ag 1.2 (agonist; lanes 3,6,9), for 4 hours (lanes 1-3), 8 hours (lanes 4-6) or 24 hours (lanes 7-9). (b) Anti-HA protein blot of cell extracts, fractionated by SDS-polyacrylamide gel electrophoresis, from cells treated with vehicle (lanes 1,4,7,10), 35 nM Hh protein (lanes 2,5,8,11) or 0.5 μM Hh-Ag 1.2 (agonist; lanes 3,6,9,12), for 2 hours (lanes 1-3), 5 hours (lanes 4-6), 8 hours (lanes 7-9) or 20 hours (lanes 10-12). (c) Anti-HA protein blot of cell extracts, fractionated by SDS-polyacrylamide gel electrophoresis, from cells treated with vehicle (lanes 1,4), 35 nM Hh protein (lanes 2,5), or 0.5 μM Hh-Ag 1.2 (agonist; lanes 3,6), for 5 hours (lanes 1-3) or 8 hours (lanes 4-6). Cells used in (c) were also treated with cycloheximide to block new protein synthesis. Blots in (b) and (c) were reprobed with anti-tubulin antibody as a sample loading control. (d) Anti-HA protein blot of cell extracts, fractionated by SDS-polyacrylamide gel electrophoresis, from cells treated with decreasing concentrations of Hh protein (lane 1, 100 nM; lane 2, 50 nM; lane 3, 25 nM; lane 4, 12.5 nM; lane 5, 6.25 nM; lane 6, 3.12 nM), or with vehicle (lane 7), or with increasing concentrations of Hh-Ag 1.2 (agonist; lane 8, 15 nM; lane 9, 31.25 nM; lane 10, 62.5 nM; lane 11, 250 nM; lane 12, 500 nM; lane 13, 1 μM) for 22 hours. All blots were visualized by autoradiography using anti-HRP (horse radish peroxidase) secondary antibodies and a chemiluminescence reagent kit (Amersham).