A nonphosphorylatable mutant of HSP27 does not localize in the nucleus and does not bind to GATA-1 to induce its degradation. (A) K562 cells transiently transfected with HA-HSP27-Wt, HA-HSP27-Ala, or a control vector, treated or not with MG132 (20μM, 5 hours), were immunoprecipitated with GATA-1 before immunoblotting with HSP27 (HA) and GATA-1 antibodies. IPCtl, immunoprecipitation with a nonrelevant antibody and G protein. Inputs: protein expression in total extracts (40 μg). (B) K562 cells were transiently transfected with HA-HSP27-Wt and HA-HSP27-Ala. Localization of the transfected HSP27 forms was determined by immunofluorescence after 24 hours of hemin treatment. (Magnification ×100; bar, 10 μm). (C) GATA-1 content was determined by Western blot in duplicate in K562 cells expressing a control vector, HA-HSP27-Wt, or HA-HSP27-Ala. (D) GATA-1 content was determined by Western blot in HeLa cells stably overexpressing HA-HSP27-Wt, HA-HSP27-Ala, or a control vector and transiently transfected with Myc-GATA-1. When indicated, cells were treated with MG132 (20μM, 5 hours). HSC70 was used as a loading control.