Deletion mutational analysis of JAZ-p53 binding. (A) A schematic representation³  of p53 deletion mutants N(1-100), M(101-308), C(309-393), N+M(1-308), M+C(101-393), p53ΔOD(1-308&365-393), and p53ΔC(30)(1-363). TAD indicates N-terminal transactivation domain; DBD, sequence-specific DNA-binding domain; OD, oligomerization domain; and RD, C-terminal regulatory domain. While ++++ and +++ represent strong or relatively strong JAZ binding, respectively, + and – stand for weak and no binding, respectively. (B-C) GST or wt and mutant GST-p53 glutathione-Sepharose beads were incubated with the cell lysate obtained from p53-null H1299 cells that transiently express FLAG-JAZ as described in “Materials and methods.” The GST or GST-p53 beads were then immunoblotted using a FLAG antibody. “20% Input” indicates 50 μg cell lysate. Bottom panels represent Fast-green staining of GST-p53 proteins (arrows). (D) A schematic representation of JAZ deletion mutants J(1-118), J(123-171), J(167-221), J(214-294), J(1-171), J(1-221), J(167-294), and J(112-294). (E) GST-JAZ (containing deletion mutants) beads were used in vitro to pull down rp53 as described in Figure 3E. Bottom panel represents Fast-green staining of GST-JAZ proteins (arrows).