Characterization of HEK293T cell line stably expressing 3xFlag-LRRK1 and LRRK2.

(A) Schematic alignment of LRRK1 and LRRK2. Predicted functional domains are drawn to scale at the relative location within the full protein sequence. For domains containing repeat sequences, predicted individual repeat units are depicted. The sequence identity and similarity for the LRR, ROC, COR and Kinase domains are given below the schematic. Also given are detailed alignments of LRRK1 and LRRK2 at the level of common LRRK2 clinical mutations. Abbreviations for the domains: ARM, armadillo repeat domain; ANK, ankyrin repeat domain; LRR, leucine rich repeat domain; ROC, Ras of comple proteins domain; COR, C-terminal of ROC domain; Kin, kinase domain; WD40, WD40 repeat domain. (B) Representative western blot analysis of HEK293T cells stably expressing (from lane 1 to 7) 3xFlag-tagged LRRK2 wild-type, T1348N GTP deficient binding mutant, K1906M kinase dead, G2019S pathogenic mutant and LRRK1 wild-type K650A GTP deficient binding mutant, K1269M kinase dead. Upper panel shows membranes probed with Flag (M2) antibody (note that LRRK2 and LRRK1 have different exposure time due to the very low expression of T1348N mutant). Lower panel shows β-tubulin loading control. (C) Representative confocal images of stable HEK293T cells expressing LRRK1 and LRRK2 wild-type and mutants. Scale bar 10 µm.