Figure 6

G2385R mutation affects LRRK2 binding properties. (A) We measured the extent of LRRK2 and SV binding by ultracentrifugation sedimentation assay. We incubated purified RFP-LRRK2 wild-type and G2385R variant with isolated SV (10 μg protein/sample). Bound RFP-LRRK2 was separated from free RFP-LRRK2 by high-speed centrifugation. We appreciated SV-bound LRRK2 by immunoblotting with anti-RFP antibody. The recovery of SV in the pellet was evaluated based on synaptophysin immunoreactivity. (B) The binding of RFP-LRRK2 wild-type and G2385R to SV was calculated as the ratio of total RFP-LRRK2 and expressed as mean ± SE; n = 6. *p < 0.05; Student’s t-test. (C) GST-pull down approach was performed to explore the interactome associated to LRRK2 WD40 domain. (Upper panels) GST-fusion proteins corresponding to WD40 domain of LRRK2 wild type and LRRK2 G2385R were used to retain interactors from adult forebrain lysate. (Lower panel) Ponceau staining shows the abundance of GST fusion proteins used as bait. (D) We evaluated the extent of synapsin I, α-tubulin, β-Actin and 14-3-3 bound to WD40 G2385R domain. Data are expressed as ratio over WD40 wild-type domain. Graph reports mean ± S.E; n = 4. *p < 0.05, Student’s T-test. (E) We isolated on streptavidin resin full-length FLAG-LRRK2 wild-type and FLAG-LRRK2 G2385R protein from N2A over-expressing cells. Interacting proteins were resolved by western-blotting. (F) We evaluated the extent of synapsin I, α-tubulin, β-Actin and 14-3-3 bound to the different LRRK2 variant. Data are expressed as the ratio over LRRK2 wild-type. Graphs report mean ± S.E; n = 4. *p < 0.05, Student’s T-test.