Fig. 2. p53 fused to the HIV-1 Rev nucleolar localization signal sequence associates with the DFC and this localization is independent of treatment with MG132. (A) Diagram of the HIV-1 Rev NoLSC'p53 in which HIV-1 Rev NoLS was tagged to the carboxyl terminus of wild-type p53. (B) H1299 cells were transfected for 24 hours with either empty vector, or vectors expressing wild-type p53 or Rev NoLSC'p53, then cells were either left untreated or treated with MG132 (10 µM) for 8 hours, solubilized in SDS electrophoresis sample buffer followed by immunoblotting with PAb DO-1, anti-HSP70, anti-nucleolin or anti-actin antibodies as a loading control. (C) H1299 cells transfected as in B were fixed with 4% paraformaldehyde, and immunostaining and visualization were performed as described in Fig. 1C. (D) Bar chart showing the percentage of cells with nucleolar localization of wild-type p53 and Rev NoLSC'p53. The data represent two independent experiments in which at least 200 cells were counted.