Figure 3.
TRIM1 ubiquitinates LRRK2 to regulate its proteasomal degradation. (a) Immunoprecipitation and ubiquitination of GFP-LRRK2 with myc-TRIM1, myc-TRIM1 C, myc-TRIM1 ΔRF, or myc-TRIM18 in the presence of HA-ubiquitin (Ub) in HEK-293T cells. The immunoblotted membrane was physically cut between LRRK2- and myc-blotted portions, with both sections additionally probed with an anti-HA primary antibody. (b) Schematic of flow cytometric assay using GFP fluorescence to measure GFP-LRRK2 turnover. Dox-inducible GFP-LRRK2 HEK-293T cells were induced for 18–24 h and transfected ("transf." in figure), dox was simultaneously withdrawn, and GFP fluorescence was measured after 18–24 h (additional validation of assay in Fig. S2). All flow cytometry ("flow cytom.") assays were performed in the dox-inducible GFP-LRRK2 HEK-293T cell lines described in Zhao et al. (2015). (c) Representative histograms of GFP-LRRK2 fluorescence in the absence or presence of dox followed by TRIM1 or empty vector transfection. (d) Quantification of GFP-LRRK2 levels 24 h after dox withdrawal in the presence of empty vector (gray bar), TRIM1 (green bar), TRIM1 ΔRF (purple bar), or TRIM1 C (orange bar). (e) Representative immunoblot of GFP-LRRK2 levels from dox-inducible HEK-293T cells in the presence of myc-TRIM1 WT, myc-TRIM1 ∆RF, or empty vector. (f) Quantification of panel e showing mean value with error bars (SEM). (g) Quantification of GFP-LRRK2 levels in the presence of chloroquine (CQ) at 25 μM for 24 h, MG132 at 2 μM for 24 h, or equivalent volume of DMSO vehicle. (h) Immunoblot of FLAG-LRRK2 levels with or without expression of myc-TRIM1 in the absence or presence of proteasomal inhibitor bortezomib (1 nM for 18 h) and in the presence of HA-ubiquitin. Bar graphs of flow cytometry assays (d and g) show normalized median green fluorescence intensity with error bars showing twice the SEM. All histograms and bar charts of flow cytometry results represent ≥10,000 single cells per condition. All coimmunoprecipitation and flow cytometry assay results show a representative experiment, with the experiment repeated a minimum of three times. Significance for flow cytometry data (d and g) was calculated using ANOVA with post hoc t test with Bonferroni correction. Significance testing for f was performed using Mann–Whitney U test. Source data are available for this figure: SourceData F3.

TRIM1 ubiquitinates LRRK2 to regulate its proteasomal degradation. (a) Immunoprecipitation and ubiquitination of GFP-LRRK2 with myc-TRIM1, myc-TRIM1 C, myc-TRIM1 ΔRF, or myc-TRIM18 in the presence of HA-ubiquitin (Ub) in HEK-293T cells. The immunoblotted membrane was physically cut between LRRK2- and myc-blotted portions, with both sections additionally probed with an anti-HA primary antibody. (b) Schematic of flow cytometric assay using GFP fluorescence to measure GFP-LRRK2 turnover. Dox-inducible GFP-LRRK2 HEK-293T cells were induced for 18–24 h and transfected ("transf." in figure), dox was simultaneously withdrawn, and GFP fluorescence was measured after 18–24 h (additional validation of assay in Fig. S2). All flow cytometry ("flow cytom.") assays were performed in the dox-inducible GFP-LRRK2 HEK-293T cell lines described in Zhao et al. (2015). (c) Representative histograms of GFP-LRRK2 fluorescence in the absence or presence of dox followed by TRIM1 or empty vector transfection. (d) Quantification of GFP-LRRK2 levels 24 h after dox withdrawal in the presence of empty vector (gray bar), TRIM1 (green bar), TRIM1 ΔRF (purple bar), or TRIM1 C (orange bar). (e) Representative immunoblot of GFP-LRRK2 levels from dox-inducible HEK-293T cells in the presence of myc-TRIM1 WT, myc-TRIM1 ∆RF, or empty vector. (f) Quantification of panel e showing mean value with error bars (SEM). (g) Quantification of GFP-LRRK2 levels in the presence of chloroquine (CQ) at 25 μM for 24 h, MG132 at 2 μM for 24 h, or equivalent volume of DMSO vehicle. (h) Immunoblot of FLAG-LRRK2 levels with or without expression of myc-TRIM1 in the absence or presence of proteasomal inhibitor bortezomib (1 nM for 18 h) and in the presence of HA-ubiquitin. Bar graphs of flow cytometry assays (d and g) show normalized median green fluorescence intensity with error bars showing twice the SEM. All histograms and bar charts of flow cytometry results represent ≥10,000 single cells per condition. All coimmunoprecipitation and flow cytometry assay results show a representative experiment, with the experiment repeated a minimum of three times. Significance for flow cytometry data (d and g) was calculated using ANOVA with post hoc t test with Bonferroni correction. Significance testing for f was performed using Mann–Whitney U test. Source data are available for this figure: SourceData F3.

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