Fig. 2

Verification of the MS results in vitro and in vivo in mouse ventral midbrain. (a-c) Western blot validation of LRRK2 binding partners using specific antibodies. ILK and GIPC1 but no other WNT/PCP signaling components interact with LRRK2 in SN4741 (a-b) and mouse ventral midbrain of E18.5 old embryos (c). b We knocked down LRRK2 using CRISPR/Cas9 methodology and generated clonal cell lines with either normal (WT) or decreased levels of LRRK2 (KD). SN4741-LRRK2-KD served as a negative control for our co-IP experiments. d-f Western blot analysis of co-IP of transiently overexpressed candidates shows that LRRK2 binds to FLOTILLIN-2 (d) and CULLIN-3 (e) in HEK293T. JIP3 (f), a previously published LRRK2 binding partner, served as positive control. g-i IF confirmed that LRRK2 co-localizes with FLOTILLIN-2 (g), CULLIN-3 (h), and JIP3 (i) in HEK293T cells. Nuclear staining by Dapi is in blue. N ≥ 3. Scale bars indicate 20 μm