Fig. 2

Phosphorylation of Thr in the p53 TXR sites is dependent on LRRK2 kinase activity. a. Interaction between LRRK2 and p53. Lysates from differentiated SH-SY5Y cell were immunoprecipitated with the indicated antibodies or control antibody, and each immunoprecipitate (p53IP, LRRK2IP or IgG IP) was subjected to the Western blot with the indicated antibodies. The experiments were carried out three times, and a representative image is shown. 20 % of input lysates are shown (Input). B & C. Phosphorylation level of Thr in the p53 TXR sites are proportional to LRRK2 expression level. Differentiated SH-SY5Y cells were transfected with the shLRRK2 (b) or myc-G2019S (c) plasmids for 18 or 24 h, respectively, and the cell lysates were immunoprecipitated with the p53 antibody. Both the immunoprecipitates (IP: p53) and 20 % or 10 % cell lysates (Input) of the shLRRK2 or G2019S transfection, respectively, were subjected to Western blot analysis with the indicated antibodies. – indicates transfection with the empty vector. D. Rat primary neurons over-expressing LRRK2 G2019S increased phosphorylation of Thr in the p53 TXR sites. The indicated cell lysates were immunoprecipitated with the p53 antibody and the immunoprecipitates (IP: p53) were subjected to Western blot. Input indicates 20 % of the cell lysates. The experiments were repeated three times, and a representative result is shown with a statistical analysis of the three results from each experiment, except D which was carried out once. Numbers below the gel figure (d) are relative protein level of each TXR band ([p-TXR]/[total p53]) based on the densitometric analysis. The antibodies used for Western blot analysis were indicated in the right side of each blot. *: p <0.05