FIGURE 5.

Mi-2 protein translation is regulated through the 5′-UTR following UV radiation. A, the 5′- and 3′-UTRs of Mi-2β were cloned into the pMIR-Report luciferase vector as depicted. B, keratinocytes were transfected with CMV-Renilla luciferase and the indicated reporter plasmid. 24 h following transfection, cells were treated with 90 J/m² UV radiation. Cell lysates were collected 2 h following treatment and analyzed for luciferase activity. Data represent the average of at least nine independent points with S.E. C, mRNA was harvested from cells transfected and treated as in B and used for real-time RT-PCR analysis. Relative luciferase message levels are graphed corrected against Renilla luciferase message. D, MCF7 cells were mock-treated or treated with 90 J/m2 UV followed by incubation with [³⁵S]methionine containing media. 2 h after treatment, cell lysates were collected and immunoprecipitated with Mi-2 specific antibodies or purified nonspecific rabbit IgG. Precipitated proteins were subjected to SDS-PAGE and visualized by phosphorimaging. The arrows mark the Mi-2 protein and a nonspecific protein used as a loading control.