5.2.8. BODIPY-Cyclopamine Binding Assay

HEK293T cells were transfected with genes encoding human WT SMO tagged with Myc-DDK or human SMO D473H mutant tagged with Myc. Cells were washed in PBS supplemented with 0.5% FBS, fixed in 4% paraformaldehyde in PBS for 10 min, and incubated for 6 h at 37 °C in the same medium supplemented with BODIPY-cyclopamine (5 nM), with and without 4s at different concentrations (1 nm at 5 µM). The cells were permeabilised with 0.2% Triton X-100 (Sigma, St. Louis, MO, USA). Dako Fluorescent Mounting solution (Dako, Carpinteria, CA, USA) was used as mounting medium and the Hoechst reagent for staining of cell nuclei. BODIPY (green) and Hoechst (blue) signals were analysed in three representative fields per coverslip (×20 magnification). Data were expressed as the percentage of BC incorporation observed with BC alone.