Endogenous interaction between VPS13C and Rab10 depends on LRRK2-mediated Rab10 phosphorylation. (A) Representative confocal images showing PLA signal (red) from VPS13C and Rab10 in control (left) and VPS13C KD (right) cells with DAPI staining (blue) (scale bar: 10 µm). (B and C) Relative quantification of PLA puncta per cell (B) and PLA area per cell (C) (N = 3, from n = 25 images [control] and n = 24 images [VPS13C KD]), confirming the interaction between VPS13C and Rab10 under physiological conditions. (D) Representative confocal images showing PLA signal (red) from MLi-2 treated control cells in comparison to vehicle (DMSO) treatment (scale bar: 10 µm). (E and F) Relative quantification of PLA punta per cell (E) and PLA area per cell (F) (N = 3, from n = 27 images), which validates the phospho-dependent interaction between VPS13C and Rab10 and indicates that the interaction depends on LRRK2-kinase activity. (G) Representative immunoblot of immunoprecipitated Rab10-GFP and co-immunoprecipitated endogenous VPS13C after treatment with MLi-2 in HEK293 FT cells. (H) Relative quantification of co-IP efficiency of VPS13C with or without MLi2 treatment (N = 3). (I) Representative confocal images showing PLA signal (red) from VPS13C and Rab10 in control (left) and VPS13C KD (right) dopaminergic neurons (scale bar: 10 µm). (J and K) Relative quantification of PLA puncta per cell (J) and PLA area per cell (K) (N = 4, from n = 30 images [control] and n = 31 images [VPS13C KD]), confirming that VPS13C and Rab10 interact in dopaminergic neurons. Data represented as mean ± SEM; unpaired two-tailed t test (B, C, E, F, H, J, and K); *P < 0.05 (H), **P < 0.01 (F), ***P < 0.001 (E), ****P < 0.0001 (B, C, J, and K). Source data are available for this figure: SourceData F5.