Figure 1.
Expression of LRRK2-GFP in U-2 OS cells via BacMam gene delivery.
U-2 OS cells were transduced with 20% BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were treated with DMSO only or LRRK2-IN-1 (3 µM) for 90 min. (A) GFP images were captured and representative images are shown. (B) Western blot analysis with anti-pSer935 antibody or anti-LRRK2 antibody. The quantifications are from 3 independent experiments.
Figure 2.
TR-FRET detection of Ser935 phosphorylation of LRRK2-GFP in U-2 OS cells.
(A) U-2 OS cells were transduced with indicated amounts of BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were plated onto a 384-well assay plate and lysed in the presence of Tb-labeled anti-pSer935 antibody. TR-FRET was measured and the emission ratios of 520 nm/490 nm are plotted against the amount of BacMam used for transduction. (B) Cells transduced with 20% BacMam LRRK2-GFP were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in (A). Emission ratios of 520 nm/495 nm are plotted against the amount of LRRK2-IN-1. All data points represent the average value (±SD) of 6 replicates.
Figure 3.
TR-FRET detection of Ser935 phosphorylation of LRRK2-GFP in SH-SY5Y cells.
(A) SH-SY5Y cells were transduced with 25% BacMam LRRK2-GFP wild-type, G2019S, R1441C or D1994A. Cells were treated with DMSO only or LRRK2-IN-1 (3 µM) for 90 min. Western blot analysis with anti-pSer935 antibody or anti-LRRK2 antibody was performed. The quantifications are from 3 independent experiments. (B) SH-SY5Y cells were transduced with indicated amounts of BacMam LRRK2-GFP wild-type, G2019S, R1441C and D1994A. Cells were plated onto a 384-well assay plate, lysed and analyzed as described in Figure 2 legend. Emission ratios of 520 nm/495 nm are plotted against the amount of BacMam used for transduction. (C) Cells transduced with 25% BacMam LRRK2-GFP were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in (B). Emission ratios of 520 nm/495 nm are plotted against the amount of LRRK2-IN-1. All data points represent the average value (±SD) of 6 replicates.
Figure 4.
TR-FRET detection of Ser935 phosphorylation of LRRK2-GFP in human neural stem cells.
Human neural stem cells were transduced with 10% BacMam LRRK2-GFP G2019S or D1994A. Cells were plated onto a 384-well assay plate and treated with indicated amounts of LRRK2-IN-1 for 90 min. Cells were lysed and analyzed as in Figure 2. Emission ratios of 520 nm/495 nm are plotted against the concentration of LRRK2-IN-1. All data points represent the average value (±SD) of 6 replicates.
Table 1.
IC50 values (µM) of known compounds determined in the TR-FRET cell-based assay for Ser935 phosphorylation of LRRK2.
Figure 5.
Tocriscreen™ mini library screening results.
Average percent inhibition of library compounds was analyzed to produce a frequency distribution with binning equal to one percent increments. The frequency distribution was plotted and a gaussian curve fit to the distribution (curve statistics: mean 4.38% standard, deviation 10.53% and R2 value of 0.962).
Table 2.
Top 16 hits of the Tocriscreen™ Mini library screen.
Figure 6.
The effects of hit compounds on LRRK2 phosphorylation at serines 910, 935, 955 and 973.
Flp-In T-REx™ HEK293 GFP-LRRK2 G2019S cells (GS) or A2016T/G2019S (IRM) were induced and treated with indicated compounds at 20 µM. After cell lysis, GFP-LRRK2 was immunoprecipitated and the phosphorylation of LRRK2 was analyzed by immunoblotting. The quantifications are from 3 independent experiments.