TRIM1 competes with 14-3-3 to bind LRRK2’s regulatory loop and recruit LRRK2 to microtubules. (a) Live-cell confocal microscopy of mCherry-LRRK2 in the presence of EBFP2-14-3-3 and GFP-TRIM1. Scale bar = 10 μM. (b) Quantification of H1299 cells with microtubule-associated LRRK2 when coexpressed with indicated proteins. 100 cells were evaluated in each experiment; bars indicate mean ± SD (two independent experiments). (c) Coimmunoprecipitation of GFP-LRRK2 WT, Ser910Ala Ser935Ala (SA), Ser910Asp Ser935Asp (SD), or R1441C (RC) with V5-14-3-3 theta in the presence and absence of myc-TRIM1 in HEK-293T cells. (d) Quantification of panel c showing mean values from three independent experiments, with error bars showing ± SD. Significance determined by Mann–Whitney U test. (e) Coimmunoprecipitation of GFP-LRRK2 with either V5-14-3-3 theta or myc-TRIM1 in HEK-293T cells. Overlaid immunoblots in color show relative ratio of phospho- to total-LRRK2 (total LRRK2 in green, antibody is NeuroMab clone N241A/34; phospho-LRRK2 is in red, antibodies are phospho-Ser910 [Abcam, UDD 1 15(3)] and phospho-Ser935 [UDD 2 10(12); Abcam]). (f) Quantification of e with mean ± SEM from three independent experiments. (g) 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. Rare cells with low levels of colocalization before treatment were followed. LRRK2 is shown in green and TRIM1 in purple. Images from isolated channels are shown in Fig. S5 c. Scale bar = 10 μM. All live-cell images and coimmunoprecipitation experiments are a representative image of at least three independent experiments. Significance testing for panel b was performed using Kruskal–Wallis with post hoc Dunn test and Bonferroni correction and Mann–Whitney U test for d and f. Source data are available for this figure: SourceData F6.