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Fig. 1. p53 localizes to the FC of the nucleolus after MG132 treatment. (A) Schematic diagram of the nucleolus with the fibrillar center (FC) in the middle, surrounded by the dense fibrillar center (DFC) and the granular components (GCs). Some nucleoli can have several FCs. (B-D) H1299 cells were transfected with wild-type p53 and after 24 hours were treated with different doses (5 µM, 10 µM and 20 µM for 6-10 hours and 1 µM, 5 µM and 10 µM for 24 hours) of MG132 for various amount of time, as indicated. Western blotting of p53 was performed using PAb DO-1 for p53, anti-nucleolin and anti-actin antibodies (B). Transfected H1299 cells as in B were fixed with 4% paraformaldehyde and then examined by immunofluorescence analysis. The cells were visualized by confocal laser scanning and DIC microscopy (C). The percentage of cells with accumulated nucleolar p53 was estimated by counting at least 200 cells in two separate experiments (D). (E) H1299 cells were transfected, treated and analyzed as describe above and immunofluorescence was performed using PAb DO-1 and PAb 1801 for p53 and anti-UBF antibodies.
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