<p><b>a.</b> Kinase activity of LRRK2 mutants tested by kinase assay using MBP as substrate. HEK293 cells were transfected with HA-tagged WT LRRK2 or mutants in the Roc-COR-Kinase domain, including R1441C, R1628P, Y1669C, I2012T, G2019S, I2020T, and kinase-inactive mutant D1994N as indicated. After 24 h, LRRK2 was immunoprecipitated 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. The bottom panel shows equal expression of HA-LRRK2 and MBP used in the lysates. <b>b.</b> The graph is the quantification of LRRK2 kinase activities in Fig 1A. The numbers are relative values, with WT set to 1. <b>c.</b> Kinase activity of LRRK2 mutants tested by kinase assay using LRRKtide as substrate. HA-tagged WT LRRK2 or mutants were immunoprecipitated as above, then <i>in vitro</i> LRRK2 kinase assay was performed using LRRKtide as substrate. <b>d.</b> Overexpression of R1628P mutation do not cause neuronal cell death. Primary-cultured cortical neurons were transfected with GFP-tagged WT LRRK2 or mutants in the Roc-COR-Kinase domain, including R1441C, R1628P, Y1669C, I2012T, G2019S, I2020T, and kinase-inactive mutant D1994N. After 48 h transfection, the dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. All the above results represent at least three independent experiments as the mean ± SD, *P<0.05, **P<0.01 (ANOVA).</p
