Ubiquitin mediates the interaction between Smo and Vps36. Ubiquitin mediates the interaction between Smo and Vps36. (A–C) S2 cells were transfected with the indicated constructs, treated with or without Hh, followed by Co-IP with anti-Myc antibody and western blot for anti-Myc to detect wild-type Smo (A,C) or Smo–Ub (B) expression, for anti-HA to detect the overexpressed Ub (A,C), for anti-V5 to detect expression of Vps36 (B,C) or for anti-FLAG to detect expression of USP8 (C) in the Co-IP product or cell lysates. Arrows indicate the bands where Vps36–V5 interacted with Myc–SmoWT or Myc–SmoWT–Ub. (A) In the absence of Hh, the ubiquitylation level of Smo was higher than that in the presence of Hh. (B) Smo–Ub interacts with Vps36 regardless of Hh. (C) USP8 decreases the ubiquitylation level of Smo and its interaction with Vps36. (D) S2 cells were transfected with the indicated constructs and treated with or without Hh, followed by immunostaining with anti-Myc antibody. Ub blocks Smo cell plasma membrane accumulation induced by Hh signal. (E–E″) A wing imaginal disc expressing UBP2 with AG4–GFP stained with anti-Smo antibody. White squares indicate the magnified regions in the A and P compartment in E″, respectively. GFP signal indicates target gene UBP2 expression regions (E–G, E″–G″). In the magnified images, expression of UBP2 accumulated Smo on the cell plasma membrane only in the A compartment (compare Smo staining in F′ and G′). Scale bars: 5 µm (F″,G″). Xiaofeng Yang et al. J Cell Sci 2013;126:4230-4238 © 2013. Published by The Company of Biologists Ltd