Fig. (1) Phosphorylation on tyrosine 327 of Tip60. (A) Rat Tip60 (isoform 1) has 546 amino acids, functional domains, several key tyrosine phosphorylation targets (Y327, Y345, and Y409), and a putative PID1 binding motif of FE65. The putative tyrosine phosphorylation site in human Tip60 was replaced with phenylalanine (Y327F, Y345F, and Y409F). Functional domains (chromodomain, Zn finger, the conserved HAT domain, and NR box) in Tip60 are indicated with amino acid number. (B) Representative immunoblot data using antibodies against phosphorylated tyrosine and Tip60. HEK293 cells were transfected with EGFP DNA constructs as indicated for immunoprecipitation with anti-GFP and immunoblotting. Expression of EGFP Tip60 protein was monitored by Western blot. Negative control (NC) was untransfected HEK293 cells. (C) GST fusion proteins of Tip60 WT or Y327F from E. coli were subjected to in vitro tyrosine kinase assay. GST fusion proteins with Tip60 WT or Y327F were monitored with Coomassie blue staining (bottom). Experiments were repeated at least three times.