Fig. 2. The C-terminal region of Hrs is required for IL-2Rβ binding. (A,C) Structures of wild-type Hrs and its deletion (d) mutants. The Vps27-Hrs-STAM (VHS), Fab1-YGL023-Vps27-EEA1 (FYVE), coiled-coil and clathrin-binding domain (CBD) are shown at the top. The ubiquitin-interacting motif (UIM), PSAP sequence, proline (Pro)-rich region and proline or glutamine (Pro/Gln)-rich region are also indicated. (B,D) Lysates from HEK293T cells (2x10⁶) cotransfected with 2 µg IL-2Rβ and 2 µg wild-type Hrs or Hrs mutants were immunoprecipitated with TU11 and immunoblotted with an anti-Hrs monoclonal antibody. The levels of IL-2β and Hrs were examined by immunoblotting with an anti-IL-2Rβ antibody (C-20) and anti-Hrs antibody, respectively, as controls. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2x10⁶) were immunoblotted with an anti-Hrs antibody. Asterisks indicate endogenous Hrs. WT, wild-type; IP, immunoprecipitation; IB, immunoblotting.