Hrs interacts with Smo and promotes Smo ubiquitination.

<p>(A) S2 cells were transfected with Myc-Smo alone or in combination with HA-Hrs or Hrs dsRNA. Cell extracts were subjected for immunoprecipitation with the anti-Myc antibody followed by western blots with anti-Ub, anti-HA, and anti-Myc to detect the ubiquitination of Smo, Smo-bound Hrs, and the levels of Myc-Smo, respectively. To normalize the levels of Smo, 50 mM MG132 and 15mM NH4Cl was used to block Smo degradation, and samples were normalized for loading. All figures in this study showing the levels of Myc-Smo were normalized by the same way. The efficiency of Hrs RNAi was determined by western blot with anti-Hrs antibody. GFP served as a transfection and loading control. (B) S2 cells were transfected with Myc-Smo alone or together with HA-Hrs, and treated with HhN-conditioned medium or control medium followed by immunoprecipitation and western blot with the indicated antibodies. Of note, the interaction between Smo and Hrs was down-regulated by Hh treatment. (C) S2 cells were transfected with Myc-Smo and treated with HhN-conditioned medium or control medium. Cell extracts were immunoprecipitated with the anti-Myc antibody and blotted with the indicated antibodies. Hh treatment reduced the interaction between Smo and endogenous Hrs.