Figure 4. LRRK2 directly interacts with NLRC4 to promote inflammasome assembly. (a) Peritoneal macrophages from WT and Lrrk2−/− mice were infected with S. Typhimurium at an MOI of 50 for 45 min. Uninfected cells were included as controls. Cell lysates were then immunoprecipitated (IP) using anti-LRRK2 antibody and were immunoblotted with the indicated antibodies. Whole-cell lysates are shown as the input. (b) LRRK2 interacts with NLRC4 but not NLRP3 and ASC. The HEK293T cells were transfected with Myc-tagged LRRK2 and cotransfected with/without Flag-tagged NLRC4, NLRP3, or ASC as indicated. Cell lysates were immunoprecipitated with anti-Flag and analyzed by immunoblot. (c) In vitro pull-down assay using recombinant LRRK2 and NLRC4 proteins: 50 ng purified 3×Flag-NLRC4 protein was incubated with 200 ng recombinant LRRK2 protein followed by immunoprecipitation with anti-LRRK2 antibody and Western blot analysis. (d) Flag-tagged WT full-length LRRK2 or different LRRK2 domains (LRR, ROC, C terminus of ROC [COR], kinase, and WD40) were coexpressed with Myc-tagged NLRC4 in 293T cells, immunoprecipitated with anti-Flag, and analyzed by immunoblot. (e) Flag-tagged full-length NLRC4 and the NLRC4 mutants ΔLRR (NLRC4 lacking LRR domain), ΔCARD (NLRC4 lacking CARD), or only nucleotide-binding domain (NBD) domain were coexpressed with Myc-tagged LRRK2 in HEK293T cells, immunoprecipitated with anti-Flag, and analyzed by immunoblot. (f) Flag-tagged NLRC4 LRR domain was coexpressed with Myc-tagged LRRK2 WD40 domain in 293T cells followed by immunoprecipitation with anti-Flag antibody and immunoblot analysis. (g) LPS-primed peritoneal macrophages from littermate control (WT) and Lrrk2−/− mice were treated with LRRK2 inhibitor GSK2578215A (GSK) at 2 µM for 1 h and then infected with S. Typhimurium at an MOI of 50 for 1 h. Cell lysates were subjected to co-immunoprecipitation with anti-LRRK2 antibody followed by Western blotting. Whole-cell lysates are shown as the input. All the results are representative of at least three independent experiments.
Figure 4.

LRRK2 directly interacts with NLRC4 to promote inflammasome assembly. (a) Peritoneal macrophages from WT and Lrrk2−/− mice were infected with S. Typhimurium at an MOI of 50 for 45 min. Uninfected cells were included as controls. Cell lysates were then immunoprecipitated (IP) using anti-LRRK2 antibody and were immunoblotted with the indicated antibodies. Whole-cell lysates are shown as the input. (b) LRRK2 interacts with NLRC4 but not NLRP3 and ASC. The HEK293T cells were transfected with Myc-tagged LRRK2 and cotransfected with/without Flag-tagged NLRC4, NLRP3, or ASC as indicated. Cell lysates were immunoprecipitated with anti-Flag and analyzed by immunoblot. (c) In vitro pull-down assay using recombinant LRRK2 and NLRC4 proteins: 50 ng purified 3×Flag-NLRC4 protein was incubated with 200 ng recombinant LRRK2 protein followed by immunoprecipitation with anti-LRRK2 antibody and Western blot analysis. (d) Flag-tagged WT full-length LRRK2 or different LRRK2 domains (LRR, ROC, C terminus of ROC [COR], kinase, and WD40) were coexpressed with Myc-tagged NLRC4 in 293T cells, immunoprecipitated with anti-Flag, and analyzed by immunoblot. (e) Flag-tagged full-length NLRC4 and the NLRC4 mutants ΔLRR (NLRC4 lacking LRR domain), ΔCARD (NLRC4 lacking CARD), or only nucleotide-binding domain (NBD) domain were coexpressed with Myc-tagged LRRK2 in HEK293T cells, immunoprecipitated with anti-Flag, and analyzed by immunoblot. (f) Flag-tagged NLRC4 LRR domain was coexpressed with Myc-tagged LRRK2 WD40 domain in 293T cells followed by immunoprecipitation with anti-Flag antibody and immunoblot analysis. (g) LPS-primed peritoneal macrophages from littermate control (WT) and Lrrk2−/− mice were treated with LRRK2 inhibitor GSK2578215A (GSK) at 2 µM for 1 h and then infected with S. Typhimurium at an MOI of 50 for 1 h. Cell lysates were subjected to co-immunoprecipitation with anti-LRRK2 antibody followed by Western blotting. Whole-cell lysates are shown as the input. All the results are representative of at least three independent experiments.

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