Trafficking of wild-type Smoothened and the M2 mutant of Smoothened. LLC-PK1 cells stably synthesizing ECFP fusion proteins with N-acetylgalactosaminyltransferase-2 (T2) and TGN38 (shown in red for better visibility) were transiently transfected with expression plasmids coding for an EYFP fusion protein with the respective Smoothened proteins (shown in green for better visibility). 3–4 d later, the cells were incubated for 5 h at 15°C before being incubated at 37°C. Immediately before (0 min) as well as 15 min (15 min) and 40 min (40 min) after shifting the cells from 15°C to 37°C, the cells were fixed and visualized in a confocal laser scanning microscope. Overlapping signals were seen between wild-type Smoothened and the two marker proteins (a–d), whereas no overlap was observed for the M2 mutant of Smoothened (e–h). Bar, 5 µm.
