Figure 4

NF-κB forms a complex at the GIS regulatory region with both wild-type p53 and p53 with a DNA-binding domain mutation. (a) p53-deficient Soas2 cells were transfected with wild-type p53 (p53WT), DNA binding domain mutant p53 (p53R175H) or vector control, and cell lysates were incubated with GIS duplexes as in Figure 2e. Proteins bound to the GIS duplex were western blotted (WB) for p53 (left panel). The right panel shows input from transfected Soas2 cell lysates. (b) p53-deficent Soas2 cells were transfected with wild-type p53 (p53WT), mutant p53R175H (p53RH) or vector control. Cell lysates were co-immunoprecipitated with anti-RELA antibody or isotypic IgG control, and western blotting was performed for p53. (c) As in Figure 2c, GIS-luciferase and TK-renilla control were transfected into p53^-/- MEF cells with or without plasmids encoding p53 (WT and RH, respectively) and RELA, and incubated with or without NFI as indicated. Firefly luciferase gene reporter activity was normalized to renilla control. Asterisks represent P < 0.05 for treatment versus control, and with inhibitor versus without inhibitor. Luciferase reporter assays are presented as mean ± standard error for at least three independent replicates.