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. 2016 Sep 19;113(40):E5866–E5875. doi: 10.1073/pnas.1606719113

Fig. 2.

Fig. 2.

The palmitoylated Shh peptide binds Ptch1. (A) Mouse Ptch1-null cells were rescued by stable expression of Ptch1-eGFP and were incubated with fluorescent Shh22-HT-TMR or Shh22C24S-HT-TMR. After washing to remove excess fluorescent protein, cells were imaged by live microscopy. The graph shows the ratio of TMR and GFP fluorescence at primary cilia. Error bars represent SE (n > 40 cilia). Representative images of primary cilia are shown under the graph. Shh22-HT-TMR binds Ptch1 at cilia in a palmitate-dependent manner. (B) As in A, but cells were incubated with Shh22-HT-TMR in the absence or the presence of 40-fold excess Shh22-HT or Shh22C24S-HT. Shh22-HT, but not Shh22C24S-HT, competes binding of Shh22-HT-TMR to Ptch1 at cilia (n > 5 cilia). (C) As in B, but with incubation with the synthetic peptides palm-Shh22 or Shh22C24S (5 μM each). Only palm-Shh22 competes Shh22-HT-TMR binding to Ptch1 (n > 5 cilia). (D) As in A, but with expression of eGFP-tagged Ptch1 or Arl13β. Shh22-HT-TMR does not bind to cilia labeled with Arl13β-eGFP (n = 20 cilia). (E) As in A, but with expression of eGFP-tagged Ptch1 or Smo. Shh22-HT-TMR does not bind to ciliary Smo-eGFP (n > 10 cilia). (F) C-terminally deleted Ptch1 (Ptch1ΔC), DispA, and NPC1 were targeted to cilia by fusion to the intracellular domain of Smo (SmoICD). (G) As in A, but with expression of eGFP-tagged Ptch1ΔC-SmoICD, DispA-SmoICD, or NPC1-SmoICD. Shh22-HT-TMR binds Ptch1ΔC, but not DispA and NPC1 (n > 10 cilia). (H) Photocrosslinking strategy to detect interaction between palmShh22 and Ptch1. The novel palmitate analog, 15-azi-palmitate, was used to synthesize the photoreactive peptide 15-azi-palm-Shh22-biotin. (I) Peptides were added to 293T cells expressing Ptch1-mCherry, and cell lysates were affinity-purified on streptavidin beads under nondenaturing conditions. Ptch1 is pulled down in a palmitate- and biotin-dependent manner. (J) NIH 3T3 cells stably expressing Ptch1-HA were incubated with 15-azi-palm-Shh22-biotin or palm-Shh22-biotin. Palm-Shh22 was used for competition. Cells were UV-irradiated, and photocrosslinking was analyzed by denaturing affinity precipitation with streptavidin. Ptch1 is specifically photocrosslinked to 15-azi-palm-Shh22-biotin.