Figure 4.
Reducing endogenous TRIM1 levels increases LRRK2 levels. (a) Relative TRIM1 mRNA expression in dox-inducible GFP-LRRK2 HEK-293T cells with dCas9 and either nontargeting sgRNA (gray bar) or four pooled TRIM1-targeting sgRNAs (red bar). Bars indicate mean ± SD. (b) Quantification of GFP-LRRK2 fluorescence with TRIM1 knocked down (red bar) compared with cells with nontargeting sgRNA (gray bar) 24 h after dox withdrawal. Error bars are twice the SEM; minimum of 10,000 live, single cells analyzed per condition. (c) Relative TRIM1 mRNA expression in Malme-3M cells with either scrambled siRNA (gray bar) or TRIM1-targeting siRNA (red bar) from the six independent experiments quantified in panel d. (d) Quantification of endogenous LRRK2 levels measured by immunoblots after TRIM1 siRNA knockdown showing mean values from six independent experiments; error bars show SEM. scr, scrambled. (e) Representative immunoblot of endogenous LRRK2 in lysate of Malme-3M cells with scrambled siRNA (left lane) or endogenous TRIM1 knocked down by targeted siRNA (right lane). (f) Immunoblot of endogenous LRRK2 in WT and TRIM1 KO HEK-293T cells. (g) Quantification of panel f showing mean value from four independent experiments, with error bars showing SEM. (h) Schematic of optical pulse-labeling experiment in which primary cortical neurons were cotransfected with mEos3.2-LRRK2 and GFP, and either TRIM1 or control plasmid. Cells were pulsed for 5–8 s with 405-nm light, causing a portion of mEos3.2-LRRK2 to fluoresce red, and cells were imaged over the indicated time period. (i) Representative primary cortical neurons from photoswitching (PS) experiment. Before photoswitching (pre-PS), mEos3.2-Red-LRRK2 was not detected. After photoswitching (post-PS) mEos3.2-Red-LRRK2 was detected, nuclear excluded (arrow), and decayed with time. LRRK2 decayed faster in neurons transfected with TRIM1. Scale bar = 10 μM. P value for b calculated using t test. Significance testing for d and g calculated using Mann–Whitney U test. Source data are available for this figure: SourceData F4.

Reducing endogenous TRIM1 levels increases LRRK2 levels. (a) Relative TRIM1 mRNA expression in dox-inducible GFP-LRRK2 HEK-293T cells with dCas9 and either nontargeting sgRNA (gray bar) or four pooled TRIM1-targeting sgRNAs (red bar). Bars indicate mean ± SD. (b) Quantification of GFP-LRRK2 fluorescence with TRIM1 knocked down (red bar) compared with cells with nontargeting sgRNA (gray bar) 24 h after dox withdrawal. Error bars are twice the SEM; minimum of 10,000 live, single cells analyzed per condition. (c) Relative TRIM1 mRNA expression in Malme-3M cells with either scrambled siRNA (gray bar) or TRIM1-targeting siRNA (red bar) from the six independent experiments quantified in panel d. (d) Quantification of endogenous LRRK2 levels measured by immunoblots after TRIM1 siRNA knockdown showing mean values from six independent experiments; error bars show SEM. scr, scrambled. (e) Representative immunoblot of endogenous LRRK2 in lysate of Malme-3M cells with scrambled siRNA (left lane) or endogenous TRIM1 knocked down by targeted siRNA (right lane). (f) Immunoblot of endogenous LRRK2 in WT and TRIM1 KO HEK-293T cells. (g) Quantification of panel f showing mean value from four independent experiments, with error bars showing SEM. (h) Schematic of optical pulse-labeling experiment in which primary cortical neurons were cotransfected with mEos3.2-LRRK2 and GFP, and either TRIM1 or control plasmid. Cells were pulsed for 5–8 s with 405-nm light, causing a portion of mEos3.2-LRRK2 to fluoresce red, and cells were imaged over the indicated time period. (i) Representative primary cortical neurons from photoswitching (PS) experiment. Before photoswitching (pre-PS), mEos3.2-Red-LRRK2 was not detected. After photoswitching (post-PS) mEos3.2-Red-LRRK2 was detected, nuclear excluded (arrow), and decayed with time. LRRK2 decayed faster in neurons transfected with TRIM1. Scale bar = 10 μM. P value for b calculated using t test. Significance testing for d and g calculated using Mann–Whitney U test. Source data are available for this figure: SourceData F4.

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