Figure 2

Protein structures verified using various p53 antibodies. (A) HeLa cells were transiently transfected with the indicated wild-type, new variant or C-truncated pSG5.HA.p53 expression DNAs (0.8 μg). Various p53 proteins were immunoprecipitated with the indicated antibodies specifically against the C-terminal (C), N-terminal (N) and conformation (Conf) regions. Immunoprecipitated extracts were subject to the western blotting analysis using the HA-tag antibody. (B) HeLa cells were transiently transfected with the indicated wild-type or new variant pSG5.HA.p53 expression DNAs (0.8 μg), and 36 hours after transfection, the immunoprecipitated lysates, by specificity for the p53 C-terminus, were subjected to the western blotting analysis using HA-tag and C-terminal antibodies. (C) HeLa cells were transiently transfected with the indicated wild-type or new variant pSG5.HA.p53 or/and Gal4DBD.p53 expression DNAs (0.5 μg), and 36 hours after transfection, the immunoprecipitated lysates, by specificity for Gal4DBD, were subjected to the western blotting analysis using HA-tag and Gal4DBD antibodies. (D) The indicated wild-type, new variant and C-terminal truncated pSG5.HA.p53 were translated in vitro and incubated with bead-bound GST protein. The various GST–p53 (wild-type and new variant) fusion proteins are indicated. Bound proteins were eluted, separated by SDS–PAGE, and visualized by autoradiography. For comparison, the leftmost lane of each panel shows 20% of the input protein used in the binding assay reactions. We observed a similar expression pattern in three independent experiments.