HSP27 induces GATA-1 ubiquitination and proteasomal degradation. (A) Recombinant GATA-1 protein, generated with TNT T7-coupled reticulocyte lysate system, was incubated in the presence or absence of recombinants ubiquitin (Ub) and HSP27 for 40 minutes in an ubiquitin buffer, as described in “In vitro ubiquitination assay.” The reaction was stopped with Laemmli buffer, run on a gel, and blotted with the Myc-tag (GATA-1) antibody. The smear corresponds to the different forms of ubiquitinated GATA-1. (B) K562 cells were transiently transfected with GATA-1 alone or GATA-1 to together with a HSP27 or an ubiquitin construct. Cells were treated 5 hours with MG132, and then lysates were immunoprecipitated with an agarose-conjugated multiubiquitin antibody followed by a Western blot with a GATA-1 antibody. Control for immunoprecipitation (IPCtl) corresponds to a mix of nonrelevant immunoglobulin G and protein G in the presence of lysate. Inputs: protein expression in total extracts (40 μg). (C) HeLa cells were transiently transfected with HA-HSP27 and Myc-GATA-1 plasmids and treated or not with MG132 (20μM, 5 hours). GATA-1 content was analyzed by immunoblot. (D) HeLa cells were transiently transfected with Myc-GATA-1 or with both Myc-GATA-1 and HA-HSP27. Cell extracts from cells either left untreated or treated with trichostatin A (300nM, 16 hours) were resolved on a gel and immunoblotted with Myc-tag (GATA-1). (E) HeLa cells were transiently transfected with Myc–GATA-1 or the acetyl mutant GATA-1 (AcMut-GATA-1) in the presence or absence of HA-HSP27. After 24 hours of transfection, expression of GATA-1 and HSP27 was assessed by Western blot. (Bottom) Densitometry analysis to quantify HSP27-induced degradation of GATA-1. One representative blot of 3 performed is shown. HSP90, HSC70, and 14-3-3 are loading controls.