Fig. 4. Hrs directly associates with IL-2Rβ in a ubiquitin-independent manner. (A) Glutathione-Sepharose beads containing immobilized GST or GST-Hrs were incubated with lysates of HEK293T cells (2x10⁶) transfected with Flag-IL-2Rβ, Flag-IL-2Rβd268-348 or Flag-IL-2Rβd349-410. Bound proteins were separated by SDS-PAGE and immunoblotted with an anti-IL-2Rβ antibody (C-20). The level of wild-type IL-2Rβ and its mutants was examined by immunoblotting with an anti-IL-2Rβ antibody (C-20) using total lysates of transfected HEK293T cells. Expression of the GST fusion proteins was detected by immunoblotting with an anti-GST monoclonal antibody. (B) HEK293T cells were cotransfected with 2 µg HA-ubiquitin (HA-UB) or the empty vector and 2 µg Flag-IL-2Rβ, Flag-IL-2Rβd268-348 or Flag-IL-2Rβd349-410. Lysates of the HEK293T cells (2x10⁶) were immunoprecipitated with TU11 and immunoblotted with an anti-HA antibody. The level of IL-2Rβ in the precipitates was examined by immunoblotting with an anti-Flag monoclonal antibody. (C) Glutathione-Sepharose beads containing immobilized GST or GST-Hrs were incubated with the His-tagged cytoplasmic tail fragment of IL-2Rβ (IL-2Rβ_269-551His). The associated proteins were analyzed by immunoblotting with an anti-IL-2Rβ antibody (C-20). The input (2%) of IL-2Rβ_269-551His was examined by immunoblotting with an anti-IL-2Rβ antibody (C-20). The levels of the GST fusion proteins were detected by western blotting with an anti-GST antibody. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2x10⁶) immunoblotted with an anti-Hrs antibody or anti-IL-2Rβ antibody. WT, wild-type; IP, immunoprecipitation; IB, immunoblotting.