Phosphorylation and 14-3-3 Binding of LRRK2 and PD Figure 1. Identification of LRRK2 phosphorylation sites in BAC transgenic brain. (A) Table of the identified phosphorylation sites within tryptic peptides from purified brain FLAG-LRRK2 protein. The estimate of the stoichiometry of phosphorylation is based on the ratio of number of MS/ MS spectra observed. The exact location of each proteolytic peptide is shown in the position column. (B) Purified brain FLAG-LRRK2 protein was treated with or without Calf-intestinal alkaline phosphatase (CIAP), followed by Western blot analysis with anti-pS935 antibody or anti-FLAG antibody. (C) FLAG-LRRK2 WT or FLAG-LRRK2 S935A mutant plasmid was transfected into HEK-293T cells; FLAG-tagged proteins were then immunoprecipitated and analyzed by anti-pS935 antibody or anti-FLAG antibody via Western blot analysis. (D) FLAG-LRRK2 was purified from different BAC-transgenic tissues and western blot was performed to detect pS935 and total FLAG-LRRK2 levels. No significant difference in the ratio of pS935/total LRRK2 was observed between different tissues. Data are presented as mean value (6 SEM) from three mice. (E) FLAG-LRRK2 was purified from 3 months and 18 months transgenic mouse brain and western blot was performed to detect pS935 and total FLAG-LRRK2 levels. No significant difference in the ratio of pS935/total LRRK2 was observed between 3 months and 18 months. The Western blot signals were quantified by using LI-COR and Odyssey software. Data are presented as mean value (6 SEM) from three mice. doi:10.1371/journal.pone.0017153.g001 results indicate that the phospho-peptide (but not the control peptide) competed for 14-3-3 binding, therefore demonstrating the critical role of pS935 in 14-3-3 binding with LRRK2. Furthermore, we co-transfected FLAG-LRRK2 with Myc-14-3- 3c wild type, mutant K50E or V181D, which was reportedly impaired in binding phospho-serine of target proteins [17,18]. In PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17153