Supplementary material for Incardona et al., Proc. Natl. Acad. Sci. USA, 10.1073/pnas.220251997

Fig. 6. Shh internalization by PtcHA is receptor mediated and dynamin dependent. PtcHA⁺ (red) colocalizing with Shh-N (green) is observed as yellow in the merged images. (A–C) A PtcHA⁺ HeLa cell overexpressing Dyn-WT incubated in 1 nM Shh-N for 2 h starting 15 min after a shift to 39°C. (D–F) A PtcHA⁺ HeLa cell overexpressing a temperature-sensitive dynamin mutant (Dyn-TS) incubated in 1 nM Shh-N for 2 h at 32°C. Note that PtcHA⁺ vesicles with internalized Shh-N appear throughout the cytoplasm. (G–I) A PtcHA⁺ HeLa cell overexpressing Dyn-TS incubated in 1 nM Shh-N for 2 h starting 15 min after a shift to 39°C. Note that Shh immunofluorescence appears predominantly near the cell surface. (J and K) Vertical optical sections of the cell shown in G–I through the region indicated by the white line in I. (J) PtcHA immunofluorescence; n, nucleus. (K) ShhN immunofluorescence. (Scale bar = 10 m M.)