Figure 1

Nutlin-induced acetylation of p53. (A) MOLM-13 cells were treated with 6 μM nutlin-3 for 0, 2, 4, 6 and 8 hours. Cells subjected to irradiation (IR) (25 Gy, 2 hours incubation) were included as a positive control, while cells treated with DMSO for 8 hours was used as a negative control. Western blotting was performed using antibodies against p53, acetylated p53 (Lys382), phoshorylated p53 (Ser15), phosphorylated p53 (Ser20), MDM2 and p21. Actin was used as loading control. (B, C) SAOS-2 and H1299 cells were transiently transfected with cDNA of p53 and treated with DMSO (control) or 6 μM nutlin-3 for 4 or 6 hours. For SAOS-2 cells, Western blotting was performed using antibodies against p53, acetylated p53 (Lys382) and actin. For H1299 cells, immunoprecipitation was performed using an anti-acetyl-lysine antibody, followed by Western blots of p53. Also Western blots of p53 and actin from total lysate used in the immunoprecipitation are shown. Bands were quantified using region of interest imaging analysis, and values are given as fold induction of control relative to actin.