Figure 6

The new p53 variant binds to ER and TR as well as wild-type p53, whereas it loses the repression on ER and TR transcriptional activities. (A and B) HeLa cells were transiently transfected with the indicated amount of the pSG5.HA.p53 expression vector; MMTV(ERE)-LUC reporter gene (0.15 μg), pHE0 (0.004 μg) encoding hERα; MMTV(TRE)-LUC reporter gene (0.15 μg), pCMX-hTRβ1 (0.004 μg) encoding hTRβ1. Transfected cultures were grown in (A) 100 nM estradiol or (B) in 100 nM T₃, and the luciferase activities of the transfected cell extracts were determined. These data are the average of three experiments (mean ± S.D.; n = 3). (C and D) The proteins indicated at the left of each panel (ER and TR) were translated in vitro and incubated with bead-bound GST fusion proteins (indicated at the top of each panel along with the indicated p53 fused to GST). The bound proteins were eluted, separated by SDS-PAGE, and visualized by autoradiography. The percentage of labeled protein bound, as determined by phosphorimager analysis, is shown below each lane. For comparison, the left lane of each panel shows the indicated percentage of the input protein used in the binding reaction. The hormone for ER, 100 nM E2, and for TR, 1 μM T3, were included in panels (C) and (D) (labeled +). We observed a similar expression pattern in two independent experiments. (E) Eluted GST and GST–p53 fusion proteins were analyzed by SDS–PAGE and Coomassie blue staining.