Figure 2.

YopM protein associates with NLRP3. (a) HEK293 cells were co-transfected with Flag-NLRP3 and Myc-YopM expression plasmids or Flag vector, and anti-Flag M2 Affinity Gel or IgG agarose immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibody. (b) Anti-Flag or IgG immunoprecipitates prepared from cells transfected with Flag-NLRP3 or Flag vector-expressing plasmids were subjected to SDS-PAGE and blotted onto nitrocellulose membrane. The nitrocellulose membrane was incubated with soluble GST-YopM (upper) or GST (middle) for 2 h and then analyzed with anti-GST or anti-Flag antibody. (c) GST-tagged YopM were subjected to pull-down assay with the lysates of HEK293 cells transfected with Flag-NLRP3 expressing plasmid. Immunoblotting analyses with anti-Flag antibody was shown in the top. Loading of the GST proteins assessed by Coomassie blue staining was shown in the bottom. GST was used as a negative control. (d) HEK293 cells were co-transfected with the indicated vector encoding Myc-tagged YopM, Flag-tagged full-length NLRP3 or truncations of NLRP3 containing only residues 1–90 (pyrin domain) (PYD), 91–710 (nucleotide-binding domain) (NBD) or 711–1033 (LRR domain) (LRR), and anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibody. All co-immunoprecipitation experiments were performed independently three times with comparable results. All cell-based experiments were performed independently three times with comparable results