Phosphorylation and 14-3-3 Binding of LRRK2 and PD Figure 3. 14-3-3 binding to LRRK2 S935 is phosphorylation-dependent. (A, B) FLAG-LRRK2 Wt or S935A mutant plasmid was transfected with or without Myc-14-3-3c into HEK-293T cells. Co-immunoprecipitation experiments were performed using either anti-myc (A) or anti-FLAG (B) antibody, followed by Western blot analysis using the indicated antibodies. (C) Sepharose bead-conjugated GST-14-3-3c was incubated with FLAG- LRRK2 transgenic brain lysate in the presence of various concentrations of phosphorylated peptide (pS935) or non-phosphorylated peptide. The binding FLAG-LRRK2 protein was pulled down and analyzed by Western blot analysis using anti-FLAG antibody. (D, E) FLAG-LRRK2 was transfected alone, with Myc-14-3-3c Wt, with Myc-14-3-3 g K50E mutant, or with Myc-14-3-3c V181D mutant into HEK-293T cells. The co-IP assay was performed using either anti-Myc (D) or anti-FLAG (E) antibody, followed by Western blot analysis using indicated antibodies. (F) FLAG-LRRK2 was transfected alone, with Myc-14-3-3c wt or Myc-14-3-3c dominant negative (DN) mutant (K50E/V181D) into HEK-293T cells. The total FLAG-LRRK2 was immunoprecipitated and analyzed by Western blot using anti-pS935 and anti-FLAG antibodies. The Western blot shows the results from three independent transfections. The quantification of the signals was done by using LI-COR imaging and Odyssey software. The data was analyzed by One- way ANOVA (** P,0.01). Data are presented as mean value (6 SEM) from three independent experiments. doi:10.1371/journal.pone.0017153.g003 PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17153