Figure 9

PFE-360 inhibits LRRK2-Ser935 and Rab10-Thr73 phosphorylation in a concentration-dependent manner in non-stimulated PBMCs from human healthy subjects. Determination of LRRK2 inhibitor IC50 values based on either Rab10-pThr73 or LRRK2-pSer935 levels in human non-stimulated PBMCs. (A) Odyssey CLx scan image showing Western Blot analysis of crude lysates from a pool of PBMCs from two donors treated for 1 hour with concentrations of PFE-360 ranging from 4nM-1µM. in duplicate. LRRK2 and Rab10 immunoreactivity (red panels), LRRK2-pSer935 and Rab10-pThr73 immunoreactivity (green panels) as well as overlay in non-stimulated human PBMCs. Full-length blots are presented in Supplementary Figure 5. Non-linear regression plot of percentage (B) LRRK2-pSer935 inhibition and (C) Rab10-pThr73 as a function of log10-transformed PFE-360 concentration. The experiment was repeated three times and the resulting IC50 determination is summarized in Table 2.