Phosphorylation and 14-3-3 Binding of LRRK2 and PD Figure 6. PKA, not LRRK2 itself, can phosphorylate S935 in vitro and within cells. (A) 0.4 mg of purified GST-LRRK2 fragments (800–1000aa) was incubated with different amount of the PKA catalytic subunit (lane 1: without PKA; lane 2: 2500 units PKA; lane 3: 10,000 units PKA). The reaction was stopped by adding 36 SDS-PAGE sample buffer and Western blot was performed by using anti-pS935 and anti-LRRK2 antibodies. (B) Different concentrations of H-89 (0, 2.5, 5 and 10 mM) were added to the PKA and GST-LRRK2 fragment reaction mixture, and Western blot analyses was performed by using anti-pS935 and anti-LRRK2 antibodies. (C) FLAG-LRRK2 was co-transfected with different amounts of PKA plasmid into HEK-293T cells (The ratio of FLAG-LRRK2 verse PKA plasmid DNA are: 0 in lane 1, 1:1 in lane 2 and 1:10 in lane 3). The transfected cell lysate was harvested and analyzed by Western blot using anti-pS935, anti-FLAG, anti-PKA, anti-pGSK and anti-b-actin antibodies. (D) FLAG-LRRK2 was transfected into HEK- 293T cells and after 40 hours transfection, 10 mM FSK was added to the cell culture medium and incubated for 30 min. Then the cells were harvested and western blot analysis was performed to study various protein levels by using anti-pS935, anti-FLAG, anti-pGSK and anti-b-actin antibodies. The pS935 LRRK2 and total LRRK2 signals were quantified by using the LI-COR Odyssey software system and the ratio of pS935 and total LRRK2 was calculated and analyzed by One-way ANOVA (** P,0.01). Western blot results of three independent experiments are shown, and data are presented as mean value (6 SEM) from three independent experiments. (E) GST-LRRK2 fragment (800–1000aa) protein was incubated with purified FLAG-LRRK2 (from mouse brains) or with PKA catalytic enzyme subunit (as positive control) (lane 1: 2500 units PKA; lane 2: 12.5 nM FLAG-LRRK2; lane 3: 25 nM FLAG-LRRK2; lane 4: 125 nM FLAG-LRRK2; lane 5: 250 nM FLAG-LRRK2) in Kinase assay buffer and western blot was performed by using anti-pS935 and anti-LRRK2 antibodies. The arrows indicated the phosphorylated form of full length LRRK2 and GST-LRRK2 fragment; the asterisks indicated the total full length LRRK2 and total GST-LRRK2 fragment, respectively. doi:10.1371/journal.pone.0017153.g006 PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e17153