Hrs UIM and N-terminal domains physically interact with Smo, but only the UIM domain is required for promoting Smo ubiquitination.

<p>(A) A schematic drawing of Hrs truncations and their ability to interact with Myc-Smo in the immunoprecipitation assay described in this Figure. (B) S2 cells were transfected with Myc-Smo and the indicated Hrs constructs followed by immunoprecipitation and western blot with the indicated antibodies. Cell lysates were also subjected to western blot to examine the expression of Hrs full-length and Hrs truncations. The bands at 25kD in the top left panel and the bands at 55kD in the top right panel indicate the IgG that served as loading control. In the last lane of top right panel, the lysate of HA-HrsNT2 was loaded in order to show the absence of the same band in the adjacent lane. (C) S2 cells were transfected with Myc-Smo and HA-Hrs constructs in combination with Flag-USP8 or USP8 RNAi to examine whether changing ubiquitination levels of Smo by USP8 could alter the interaction between Smo and Hrs. Cell extracts were immunoprecipitated by the anti-Myc antibody followed by western blot with either the anti-HA or the anti-Myc antibody to examine the amount of Smo-bound Hrs and the levels of Smo. Western blot of the cell lysates was to examine the expression of Hrs or USP8. (D) S2 cells were co-transfected with HA-HrsNT1 that contains the UIM domain and Myc-Smo or Myc-SmoK8R that bears K>R mutation in the domain binding Hrs. The immunoprecipitation assay was performed with the anti-Myc antibody and the Smo-bound Hrs was examined by the anti-HA antibody. K>R mutation did not change the interaction between Smo and HrsNT1. (E) S2 cells were transfected with the indicated constructs or Hrs dsRNA. Cell extracts were subjected to immunoprecipitation with the anti-Myc antibody followed by western blot with the anti-Myc or anti-Flag antibodies. Cell lysates were subjected to direct western blot with the anti-Flag and anti-HA antibodies to examine the protein expressed. (F) S2 cells were transfected with Myc-Smo and Hrs variants to examine the ability of different forms of Hrs in regulating Smo ubiquitination.