Figure 9. TRIM1 mediates proteasomal... | Rockefeller University Press

Figure 9.

$TRIM1 mediates proteasomal degradation of PD-mutant LRRK2 G2019S to rescue its toxicity. (a) Live-cell confocal microscopy of GFP-LRRK2 G2019S and mCherry-TRIM1 transiently transfected into H1299 cells. Scale bar = 10 μM. (b) Coimmunoprecipitation and ubiquitination (Ub) of GFP-LRRK2 G2019S with myc-TRIM1 in the presence of HA-ubiquitin in HEK-293T cells. (c) Flow cytometric assay on dox-inducible GFP-LRRK2 G2019S HEK-293T cells in the presence and absence of TRIM1 and the proteasome inhibitor MG132; bars show median green fluorescence intensity with error bars showing twice the SEM. (d) Representative dox-inducible LRRK2 G2019S PC-12 cells transfected with mCherry-TRIM1 or mCherry alone vector and GFP and differentiated with NGF for 5 d in the presence and absence of 1 µg/ml dox. Scale bar = 10 μM. (e) Quantification of the fraction of neurite-bearing PC-12 cells in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show mean of three independent experiments of 150–250 cells each; error bars show SEM. (f) Quantification of average neurite length on PC-12 cells with neurites in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show mean of three independent experiments; error bars show SEM. Significance testing for panel c was performed using ANOVA with post hoc t test with Bonferroni correction. Significance testing for panel e was performed using a test for equality of binomial parameters, and for panel f, using Kruskal–Wallis with post hoc Dunn test and Bonferroni correction. Source data are available for this figure: SourceData F9.$

TRIM1 mediates proteasomal degradation of PD-mutant LRRK2 G2019S to rescue its toxicity. (a) Live-cell confocal microscopy of GFP-LRRK2 G2019S and mCherry-TRIM1 transiently transfected into H1299 cells. Scale bar = 10 μM. (b) Coimmunoprecipitation and ubiquitination (Ub) of GFP-LRRK2 G2019S with myc-TRIM1 in the presence of HA-ubiquitin in HEK-293T cells. (c) Flow cytometric assay on dox-inducible GFP-LRRK2 G2019S HEK-293T cells in the presence and absence of TRIM1 and the proteasome inhibitor MG132; bars show median green fluorescence intensity with error bars showing twice the SEM. (d) Representative dox-inducible LRRK2 G2019S PC-12 cells transfected with mCherry-TRIM1 or mCherry alone vector and GFP and differentiated with NGF for 5 d in the presence and absence of 1 µg/ml dox. Scale bar = 10 μM. (e) Quantification of the fraction of neurite-bearing PC-12 cells in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show mean of three independent experiments of 150–250 cells each; error bars show SEM. (f) Quantification of average neurite length on PC-12 cells with neurites in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show mean of three independent experiments; error bars show SEM. Significance testing for panel c was performed using ANOVA with post hoc t test with Bonferroni correction. Significance testing for panel e was performed using a test for equality of binomial parameters, and for panel f, using Kruskal–Wallis with post hoc Dunn test and Bonferroni correction. Source data are available for this figure: SourceData F9.