Figure 5: The ATF3–Tip60 interaction is required for Tip60 regulation.

(a) GST-pulldown assays defined Tip60 regions for ATF3 binding. One microgram of indicated Tip60-truncated proteins fused to GST were immobilized on glutathione agarose and incubated with 5 μl of in vitro-translated ATF3 for GST-pulldown assays. The pulldown proteins were detected by western blotting using the ATF3 antibody. Ponceau S staining of the blot is shown in Supplementary Fig. 5. (b) GST-pulldown assays defined ATF3 regions required for Tip60 binding. Indicated ATF3 fragments (1 μg) were fused with GST, and were incubated with [³⁵S]-labelled Tip60 for GST-pulldown assays. (c) The Tip60-binding-deficient mutant (ΔATF3) lost the activity to promote Tip60 autoacetylation. The experiments were carried out as described in Fig. 2b. (d) ΔATF3 failed to increase the Tip60 expression level. H1299 cells were transfected with 0.4 μg of Tip60, and 1.6 ATF3 or ΔATF3 as indicated for western blotting. (e) ΔATF3 did not decrease the Tip60 ubiquitination level as shown by western blotting. The experiments were carried out as described in Fig. 3e. (f) Recombinant ΔATF3 failed to promote USP7-mediated deubiquitination of Tip60 as determined by in vitro deubiquitination assays as in Fig. 4b.