<p><b>a.</b> Plasmids of HA-tagged wild-type (WT) or R1628P mutant LRRK2 were cotransfected with Cdk5/p35 in HEK293 cells, as indicated. After 24 h, LRRK2 was immune precipitated in the lysates by anti-HA antibody, and <i>in vitro</i> LRRK2 kinase assay was performed to measure the LRRK2 kinas activity using purified MBP protein as substrate. HA-LRRK2, Cdk5, and p35 levels were determined by Western blotting as a loading control. <b>b.</b> The graph is the quantification of LRRK2 kinase activities in Fig 3A. The numbers are relative values, with WT w/o Cdk5/p35 set to 1. <b>c.</b> Plasmids of HA-tagged wild-type (WT), R1628P, and S1627A:R1628P mutant LRRK2 were cotransfected with Cdk5 or dominant-negative form of Cdk5 (dnCdk5) and p35 in HEK293 cells, as indicated. After 24 h, LRRK2 was immune precipitated in the lysates by anti-HA antibody, and <i>in vitro</i> LRRK2 kinase activity was measured for 30min using LRRKtide as substrate. The results represent as the mean ± SD, **P<0.01 (compared with WT:control group) ##P<0.01 (compared with WT:Cdk5/p35 group) (ANOVA). <b>d.</b> Phosphorylation mimic of S1627 (S1627D) increased the LRRK2 kinase activity. HEK293 cells were transfected with HA-tagged LRRK2 plasmids, including wild-type (WT), R1628P, S1627D and S1627D:R1628P double mutant. LRRK2 kinase activities were measured as above. <b>e.</b> The graph is the quantification of LRRK2 kinase activities in Fig 3D. The numbers are relative values, with WT set to 1. **P<0.01 (ANOVA) <b>f.</b> HEK293 cells were transfected with HA-tagged LRRK2 plasmids, including wild-type (WT), R1628P, S1627D and S1627D:R1628P. LRRK2 kinase activities were measured as above for indicated period of time using LRRKtide as substrate. **P<0.01 (compared with WT group, ANOVA)</p
