HSP27 phosphorylation is required for its association with GATA-1 and to induce GATA-1 degradation. (A) Human primary CD36⁺ erythroblasts were induced to differentiate in the presence of IL-3, and Epo. Phosphorylated HSP27 expression (on S15) was assessed at the indicated days by Western blotting. Actin serves as a loading control. (B) Localization of S15-phosphorylated HSP27 during Epo-induced CD36⁺ differentiation. Immunofluorescence was performed as described in “Methods.” Nuclei were stained with Hoechst 33342 (1 μg/mL). Magnification ×40; bar, 10 μm. (C) Phosphorylated HSP27 (on S15) was determined by Western blot in K562 erythroid cells treated with hemin. HSC70 serves as loading control. (D) Localization by immunofluorescence studies of S15-phosphorylated HSP27 during hemin-induced K562 differentiation (days 0-2). Magnification ×40; bar, 10 μm. Arrows indicate nuclear localization of phosphorylated HSP27 (E) Phosphorylated HSP27 (both on S15 and S78) was determined in lysates from K562 cells induced to differentiate with hemin (day 0 to day 3) in the presence or absence of the p38MAPK pathway inhibitor SB-203580 (20μM). (F) GATA-1 level was determined in lysates from K562 cells induced to differentiate with hemin (day 0 to day 2) in the presence or absence of the p38MAPK pathway inhibitor SB-203580 (20μM). (G) At the indicated days after hemin treatment of K562 cells, GATA-1 was immunoprecipitated followed by immunoblotting with P-Ser15-HSP27 and P-Ser78-HSP27 antibodies. IPCtl, immunoprecipitation with a nonrelevant antibody and G protein. HSP90 serves as a loading control. Inputs: protein expression in total extracts (40 μg). DMSO indicates dimethyl sulfoxide.