Figure 7. In vitro interaction of Zhangfei and p53. (A) Zhangfei associates with p53 in U2OS cells. Cell lysate from U2OS and MG63 human cells previously infected with adenovirus vector expressing Zhangfei (Ad-ZF) or mock infected (MI) were incubated with 2 µg of mouse anti-p53 antibody for 12 h at 4 °C, followed by an additional 4 h incubation with 100 µl of Protein A/G agarose beads (IP). The associated proteins were detected by immunoblotting (IB) using rabbit anti-Zhangfei antibody and mouse anti-p53 antibody. Input represented 1/10 of lysate used for immunoprecipitation. (B) Zhangfei displaces Mdm2 from p53 in U2OS cells. Cell lysates as in (A) were immunoprecipitated with anti-p53 antibody followed by immunoblotting with either anti-Mdm2 or anti-Zhangfei antibody. (C) Inhibition of proteasomal degradation reverses degradation of p53 in the absence of Zhangfei. Cells infected as in (A) were either treated with the proteasomal inhibitor MG132 (5 µM) or left untreated. Proteins in the cell lysates were separated by SDS-PAGE, and p53, Zhangfei and GAPDH (loading control) were detected by immunoblotting. (D) Schematic diagram of a model for the proposed mechanism by which Zhangfei inhibits cell growth and UPR. Under normal conditions, Mdm2 translocates the p53 protein out of the nucleus for degradation via the ubiquitin-dependent pathway. In Zhangfei-expressing cells, Zhangfei interacts with p53 and prevents it from binding to Mdm2. This results in p53 stabilization and nuclear accumulation, which, in turn, activates its target genes and suppresses tumor cell growth and the UPR. These results illustrate how Zhangfei controls the activity of p53 toward the cell growth and the UPR, and offer an explanation to why p53 is upregulated by Zhangfei in tumor cells.