Figure 3. LRRK2 promotes ASC speck... | Rockefeller University Press

$Figure 3. LRRK2 promotes ASC speck formation and assembly of NLRC4 inflammasome. (a) Peritoneal macrophages from littermate control (WT) and Lrrk2−/− mice were infected with S. Typhimurium at an MOI of 50 for 1 or 2 h. After cross-linking with DTBP, cell lysates were immunoprecipitated (IP) with anti-ASC antibody and immunoblotted with the indicated antibodies. Whole-cell lysates are shown as the input. (b and c) LPS-primed WT and Lrrk2−/− peritoneal macrophages were either infected with S. Typhimurium at an MOI of 50 (b) or cytosolic delivery of 1 µg/ml LFn-flagellin (c). ASC speck formation was assayed by ASC immunofluorescent staining, and cells were counterstained by DAPI (blue). Fluorescent images were analyzed by confocal microscopy. Percentages of macrophages containing ASC foci was quantified (right), with at least 200 cells counted in each experiment. Each condition was performed in triplicate. Quantitative data are shown as means ± SEM; ***, P < 0.001; determined by Student’s test. Arrowheads mark ASC specks. Bars, 10 µm. (d) Peritoneal macrophages from littermate control (WT) and Lrrk2−/− mice were infected with S. Typhimurium at an MOI of 50 for 1 h. Cells were dissolved with Triton X-100–containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Immunoblots of those insoluble fractions (Insoluble + DSS) and soluble fractions were detected with an antibody to ASC. (e) Immunoblot analysis of ASC in cross-linked Triton X-100–insoluble fractions from LPS-primed WT and Lrrk2−/− macrophages treated with 1 µg/ml LFn-flagellin + anthrax-protective antigen (PA) for 30 min. All results are representative of three independent experiments.$

Figure 3.

LRRK2 promotes ASC speck formation and assembly of NLRC4 inflammasome. (a) Peritoneal macrophages from littermate control (WT) and Lrrk2^−/− mice were infected with S. Typhimurium at an MOI of 50 for 1 or 2 h. After cross-linking with DTBP, cell lysates were immunoprecipitated (IP) with anti-ASC antibody and immunoblotted with the indicated antibodies. Whole-cell lysates are shown as the input. (b and c) LPS-primed WT and Lrrk2^−/− peritoneal macrophages were either infected with S. Typhimurium at an MOI of 50 (b) or cytosolic delivery of 1 µg/ml LFn-flagellin (c). ASC speck formation was assayed by ASC immunofluorescent staining, and cells were counterstained by DAPI (blue). Fluorescent images were analyzed by confocal microscopy. Percentages of macrophages containing ASC foci was quantified (right), with at least 200 cells counted in each experiment. Each condition was performed in triplicate. Quantitative data are shown as means ± SEM; ***, P < 0.001; determined by Student’s test. Arrowheads mark ASC specks. Bars, 10 µm. (d) Peritoneal macrophages from littermate control (WT) and Lrrk2^−/− mice were infected with S. Typhimurium at an MOI of 50 for 1 h. Cells were dissolved with Triton X-100–containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Immunoblots of those insoluble fractions (Insoluble + DSS) and soluble fractions were detected with an antibody to ASC. (e) Immunoblot analysis of ASC in cross-linked Triton X-100–insoluble fractions from LPS-primed WT and Lrrk2^−/− macrophages treated with 1 µg/ml LFn-flagellin + anthrax-protective antigen (PA) for 30 min. All results are representative of three independent experiments.

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