Phosphorylation and 14-3-3 Binding of LRRK2 and PD Figure 5. PD-linked mutants affect S935 phosphorylation and 14-3-3 binding of LRRK2. (A) FLAG-LRRK2 WT, S935A, R1441G, Y1699C, G2019S or K1906M mutant plasmid was transfected into HEK-293T cells. The LRRK2 variants were immunoprecipitated and analyzed by Western blot using anti-pS935 and anti-FLAG antibodies. Quantification of the signals was performed using LI-COR and Odyssey software and the data was analyzed by One-way ANOVA (** P,0.01). Data are presented as mean value (6 SEM) from three independent experiments. (B) FLAG-LRRK2-WT, G2019S, R1441G and K1906M was immunoprecipitated from the brain lysates of the corresponding BAC-transgenic mice. The quantification of the signals was performed as described above. The ratio of pS935 signal over total LRRK2 signal is shown. The data was analyzed with One-way ANOVA (* P,0.05, ** P,0.01). Data are presented as mean value (6 SEM) from three mice. (C) Myc-14-3-3c (or control Myc vector) was co-transfected with FLAG-LRRK2 Wt, S935A, R1441G or G2019S mutant plasmid into HEK-293T cells. Co-IP was performed by using anti-Myc antibody. The pulled down PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17153