Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

Figure 3

TCP80 and RHA interact in vivo and cooperatively stimulate p53 expression in MCF-7 cells. (a) TCP80 has increased binding with RHA following DNA damage in MCF-7 cells. Subconfluent MCF-7 cells were treated with or without 10 μM etoposide for 2 hours and then lysed with TGN buffer [8]. RHA was immunoprecipitated from the cell lysate as described in experimental procedures. The precipitated beads were then washed three times with TGN lysis buffer and SDS sample loading buffer was added. The samples were subjected to SDS-PAGE. An immunoblotting experiment was then performed to detect the TCP80 protein. The results presented are representative of three individual experiments. (b) Levels of TCP80 and RHA protein do not change following exposure to DNA damage in MCF-7 cells. MCF-7 cells were treated with 10 μM etoposide for 2 hours and then lysed with TGN lysis buffer. The samples were subjected to SDS-PAGE. TCP80, RHA, and β-actin were detected by their respective antibodies. The results presented in (a) and (b) are representative of three individual experiments. (c) Overexpression of TCP80 and RHA leads to increased p53 expression in H1299 cells transfected with the pC53-SN3 vector. H1299 lung carcinoma cells (p53-null) were cotransfected with the p53 expression vector pC53-SN3 along with the empty pCDNA 3.1 vector, the TCP80 expression vector, or the TCP80 plus RHA expression vector. Twenty-four hours after transfection, the cells were treated with or without etoposide for 2 hours. Cells were then lysed, and equal amounts of protein were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The p53 protein and β-actin were then detected by their respective antibodies. (d) Statistical analysis of the expression levels of p53 (p53/β-actin) between individual groups as shown in (c) was performed using one-way ANOVA with a Newman–Keul post hoc test from 4 sets of experimental results. Significance was assumed at .

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