FIGURE 3.

Acetylation of Lys⁵⁶⁴ just after the CtBP-binding consensus sequences is important for transcriptional activation of GATA2. A, various FLAG-tagged EVI1 deletion mutants (illustrated in Fig. 3A) were co-transfected with the GATA2-luciferase promoter/reporter construct into COS7 cells, and the luciferase activity was assayed. Three EVI1 mutants with lysine to alanine replacements were also used as follows: EVI1 K559A, K561A, and K564A. B, protein expression of the various kinds of transfected EVI1 deletion mutants was confirmed by immunoblotting using an anti-FLAG antibody. WT, wild type. C, characterization of anti-acetylated Lys⁵⁶⁴ EVI1 antibody made by immunization with anti-acetylated EVI1 peptides. EVI1 with K561A or K564A mutations was transfected into COS7 cells, and the cell lysates were immunoblotted with anti-FLAG antibody (α-FLAG) or anti-acetylated Lys⁵⁶⁴ EVI1 antibody (α-AcK⁵⁶⁴ EVI1). D, histochemical staining of EVI1 with anti-EVI1 or anti-AcK⁵⁶⁴ EVI1 antibody in NT2 neuronal cells, UCSD/AML1 leukemia cells, and CD34⁺ cord blood cells (CD34⁺ CBC). Each cell was stained with 4′,6-diamino-2-phenylindole or TOTO-3 for the nucleus (blue), anti-EVI1 antibody (Alexa Fluor 488, green), or anti-AcK⁵⁶⁴ EVI1 antibody (Alexa Fluor 555, red).