Figure 4

K921 Abl acetylation is mediated by Tip60 acetyl-transferase. (a) Human colorectal cancer cells (HCT116+Ch3;H3) were transfected with siRNA specific for Luciferase, Tip60 or hMOF and then mock treated or exposed to IR (5 Gy) at 36 h post-transfection. Lysates were formed at 4 post-treatment, normalized for equal protein content, and immune-analyzed with K921 Ac Abl or Abl antibody. An aliquot of the lysate was also subjected to immunoblotting with anti-Tip60 or hMOF antibody. (b) 293T cells were transfected with plasmids carrying WT or HAT-defective Tip60mt cDNA, and at 36 h post-transfection, cells were mock-treated or exposed to 5 Gy of IR. Lysates were formed 4 h later and subjected to immunoblotting with K921 Ac Abl (top panel), anti-Flag (middle panel) and anti-Tip60 antibodies, respectively (bottom panel). (c) Structural domains of GST-Abl fusion products (Abl-ABC, Abl-XS and Abl-SE). (d) Tip60 acetylates c-Abl in vitro. GST-fusion proteins, shown in panel C, were expressed in bacteria purified, and incubated with Tip60 immunoprecipitates prepared from IR (5 Gy)-treated HeLa cells in the presence of ¹⁴C-acetyl CoA in HAT buffer. The reaction products were resolved in 5-15% SDS-PAGE and the gel was dried and exposed to autoradiography (top panel). Coomassie blue staining of the GST-fusion products (middle panel). Anti-Tip60 immunoblotting (bottom panel).