Fig. 3

LRRK2 kinase activity regulates nuclear localization of p53. Down-regulation of LRRK2 expression by shLRRK2 transfection (a) or over-expressing G2019S (b) regulates the amount of nuclear p53. Differentiated SH-SY5Y cells were transfected with the indicated plasmids, the cell lysates were fractionated, and the cytoplasmic and nuclear fractions were subjected to the Western blot with the indicated antibodies. Lamin B and HSP90 were used for nuclear and cytoplasmic markers, respectively. (c) Nuclear localization of p53 protein was increased in the phosphomimetic T304/377D mutant compared to p53 WT. HA-tagged p53 WT or T304/377D plasmids were transfected to the SH-SY5Y cells, and the cells were immunostained with DAPI and Alexa 488 (HA). Ten images were taken and the transfected cells were chosen for the analysis (n = 3, Total cell numbers are 143 for the WT and 118 for the mutant). The localization of p53 protein was blindly determined as nuclear, cytoplasmic, or both, and the number of cells of each case was counted. Each representative image and the bar graph are shown. # and * indicate cells predominantly expressing cytoplasmic and nuclear p53, respectively. *: p <0.05