Evaluation of LRRK2 levels and validation of flow cytometric system to measure LRRK2 turnover. (a) Immunoblot of FLAG-LRRK2 cotransfected with myc-TRIM1 or empty vector control. Time indicates hours after transfection. (b) Quantification of panel a with LRRK2 levels normalized to actin. (c) Immunoblot showing LRRK2 levels relative to actin after withdrawal of dox (dox-induced for 18 h). (d) Histograms of GFP fluorescence from samples immunoblotted in panel c. (e) Immunoblot of dox-induced GFP expression cotransfected with myc-TRIM1 or empty vector control. Time indicates hours after transfection. (f) Flow cytometric quantification of GFP-LRRK2 levels in the presence of TRIM1, CHIP, or TRIM18. Bars show median green fluorescence intensity, with error bars showing twice the SEM. (g) Flow cytometric quantification of GFP-LRRK2 levels in TRIM1 knockdown and control dCas9/dox-GFP-LRRK2 HEK-293T lines 0, 4, 24, and 44 h after dox withdrawal relative to 0 h. Bars show median green fluorescence intensity, with error bars showing twice the SEM. Significance testing for f and g was performed using ANOVA with post hoc t test with Bonferroni correction. Source data are available for this figure: SourceData FS2.