Additional c haracterization of effect of TRIM1 on LRRK2 localization and function. (a) Live-cell confocal microscopy of GFP-LRRK2 in the presence of mCherry-TRIM1 after treatment with LRRK2 kinase inhibitor MLi-2 (200 nM) or vehicle, showing the individual mCherry and GFP channels from the time course in Fig. 6 g. Rare cells with low levels of colocalization before treatment were followed over time. Scale bar = 10 μM. (b) Quantification of GFP-LRRK2 fluorescence in flow cytometric assay with TRIM1 knocked down (red bar) compared with cells with nontargeting sgRNA (gray bar). Cells were dox-induced for 24 h, dox was removed, and MLi-2 (100 nM) or vehicle was added for another 24 h before cells were assayed. Bars show median green fluorescence intensity, with error bars showing twice the SEM. (c) Immunoblot of Rab29 phosphorylation in the presence and absence of TRIM1 for WT LRRK2 and LRRK2 K831R. (d) Quantification of Rab29 phosphorylation in panel c. (e) Live-cell confocal microscopy of GFP-LRRK2 G2019S and mCherry-TRIM1 transiently transfected into PC-12 cells (scale bar = 5 μM). From top to bottom: GFP-LRRK2 G2019S, mCherry-TRIM1, merged image, and higher magnification of area in yellow boxes. (f) Immunoprecipitation and ubiquitination of GFP-LRRK2 with myc-TRIM1 in the presence of HA-ubiquitin in PC12 cells. The immunoblot membrane was physically cut between LRRK2- and myc-blotted portions, with both sections additionally probed with an anti-ubiquitin primary antibody. Significance testing for panel b was performed using ANOVA with post hoc t test with Bonferroni correction, and for panel d, using Mann–Whitney U test. Source data are available for this figure: SourceData FS5.