Figure 3

ATM phosphorylated Abl is targeted for acetyl-modification on K921. (a) HeLa cells were incubated with STI571 (10 μM; 1h) and then either mock treated or exposed to 5 Gy of IR. At 4 h post-treatment, lysates were prepared, adjusted for equal protein content and immune-analyzed with K921 Ac-Abl antibody (top panel) or K-12 Abl antibody (middle panel). Anti-Abl immunoprecipitates were also prepared and probed with anti-pTyr antibody (bottom panel). (b) IR-induced acetylation of WT and Kinase-defective Abl mutant. Abl-null 3T3s reconstituted with WT or kinase-defective Abl (K290R) were exposed to IR (5 Gy) and lysates were formed and subjected to immunoblot analysis with K921 Ac-Abl antibody. (c) Mock- and IR-treated ATM-deficient(EBS-7) and ATM-proficient (YZ-5) fibroblasts were collected at 4 h post-treatment and lysates were formed, adjusted for equal protein concentration, and subjected to immunoblotting with K921 Ac Abl (top) or anti-Abl (bottom) antibody. (d) 293T cells were transfected with WT, S465A or S465E mutant of Abl cDNA. At 36 h post-transfection, cells were exposed to IR (5 Gy) and lysates were formed 4 h later and subjected to immunoblotting with K921 Ac Abl (top panel) or anti-Flag (middle panel) or anti-pTyr (4G10) antibody (bottom panel). (e) Fold increase in acetylation was assessed by densitometric analysis of the immune-reactive band using phosphor-imager and the data are presented as means ± SD, n = 3.