Fig. 1. Hrs associates with IL-2Rβ lacking the UIM domain. (A) HEK293T cells were cotransfected with 2 µg wild-type Flag-IL-2Rβ, Flag-IL-2R or the empty vector and 2 µg wild-type Hrs. Lysates of the HEK293T cells (2x10⁶) were immunoprecipitated with an anti-Flag monoclonal antibody and immunoblotted with an anti-Hrs monoclonal antibody. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2x10⁶) were immunoblotted with an anti-Hrs antibody. (B) Lysates of HEK293T and HEK293Tβ4 cells (5x10⁷) were immunoprecipitated with an anti-IL-2Rβ monoclonal antibody (TU11) and immunoblotted with an anti-Hrs antibody. Total lysate: aliquots (0.05%) of lysates from the indicated cells (5x10⁷) were immunoblotted with an anti-Hrs antibody. (C) HEK293T cells were cotransfected with 2 µg wild-type Flag-IL-2Rβ and 2 µg wild-type Hrs, Hrsd257-277 (dUIM) mutant or the empty vector. Lysates of the HEK293T cells (2x10⁶) were immunoprecipitated with TU11 and immunoblotted with an anti-Hrs antibody. The levels of IL-2Rβ and IL-2Rc in the precipitates were examined by immunoblotting with an anti-Flag antibody, whereas the levels of Hrs in the total lysates of transfected HEK293T cells were examined by immunoblotting with an anti-Hrs antibody. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2x10⁶) were immunoblotted with an anti-Hrs antibody. WT, wild-type; IP, immunoprecipitation; IB, immunoblotting.