Pak1ip1 localizes to the nucleolus and its mutation increases Tp53 levels.

<p>Intracellular distribution of Pak1ip1 revealed through immunofluorescence in cultured primary fibroblasts derived from wild-type (<b>A–C</b>) and homozygous mutant embryo (<b>D–F</b>). Fluorescent labeling for Pak1ip1 (<b>A, D</b>), nucleolin (<b>B, E</b>), and merged colors with DAPI (<b>C, F</b>) show an exclusively nucleolar localization in wild-type (<b>A–C</b>) and mutant cells (<b>D–F</b>) perfectly overlapping with the nucleolin signal. Scale bar is 10 μm. The western blot analysis in <b>G</b> performed on total lysate form E10.5 embryos reveals an upregulation of Tp53 in <i>mray/mray</i> mutants over wild-type littermates. The graph in <b>H</b> shows the results of the quantitative and statistical analysis of relative band intensity compared to background for wild-type (black) and <i>mray/mray</i> (red) samples. Dots correspond to individual data points, the thick lines show the mean, and the thin bars the standard error within the sample sets. A Student's t-test was used to assign significance to the data; a p-value <0.05 was obtained from the analysis. In <b>I</b>, the results of a Mdm2 co-IP are shown, which was used to pull Pak1ip1 protein from E13.5 embryo lysates. No differential signal intensity can be detected between wild-type and homozygous mutants for the subsequent western analysis of Pak1ip1.