Interaction of SHD1 and STAT5. (A,B) In vivo binding of SHD1 and STAT5. Plasmids expressing HA-tagged SHD1 and STAT5A were transfected into 293T cells, and the cell lysates were immunoprecipitated with either anti-HA (A) or anti-STAT5 (B) antibodies. The precipitated proteins were detected by Western blotting using the same sets of antibodies. (C) The in vitro binding of purified SHD1 and in vitro–translated STAT5. In vitro–translated STAT5A or STAT5B labeled with ³⁵S-methionine was mixed with GST-SHD1 in binding buffer, and pulled down with glutathione sepharose beads. The pulled down proteins were separated on SDS-PAGE gel, which was dried and exposed on x-ray film. (D) GST or GST-SHD1 protein was mixed with the lysates of starved BaF3 cells or cells stimulated with IL-3 (25 U/mL) for 10 minutes, and pulled down with glutathione sepharose beads. Immunoprecipitation using anti-STAT5 antibody was performed as a control. The precipitated proteins were analyzed by SDS-PAGE and Western blotting using anti-STAT5 antibody. (E) Association of endogenous SHD1 and STAT5. STAT5 was immunoprecipitated from the nuclear extracts of BaF3 cells using anti-STAT5 antibody or control IgG. The precipitated proteins were analyzed by SDS-PAGE and Western blotting using anti-SHD1 or anti-STAT5 antibody. (F) Analysis of SHD1-interacting domain in STAT5B. A variety of deletion mutants of STAT5B were assessed for their ability to interact with SHD1 by yeast 2-hybrid assay. The numbers denote the position of the amino acids of STAT5B. The interaction is presented as the β-gal activity. N-ter indicates N-terminus; DBD, DNA-binding domain; SH2, Src-homology 2 domain; and AD, activation domain. (G) An analysis of STAT5 interacting domain in SHD1. Various mutants of SHD1-S were assessed for their ability to interact with STAT5B by yeast 2-hybrid assay similarly as in panel F. Numbers denote the position of amino acids of SHD1-S. In (LL-AA) mutants, LXXLL motif was mutated to LXXAA.