Fig. 3.
RKIP directly interacts with ASC to inhibit inflammasome activation. a Myc-RKIP was cotransfected with Mock, NLRP1, AIM2, NLRP3, NLRC4, ASC, or pro-caspase-1 in 293T cells (4 × 105). Cell lysates were immunoprecipitated with anti-Flag beads (M2 beads) after 48 h, and then, immunoblotting analysis of the samples was performed as indicated. The star indicates the target bands. A total of 8 × 106 THP-1 stable transfectants (which stably express Flag-RKIP) were differentiated with 100 nM PMA overnight in 6 cm dishes and then primed with LPS and treated with nigericin (Ni) and flagellin (b) or poly(dA:dT) (c). Whole cell lysates were immunoprecipitated with anti-Flag or control IgG, followed by immunoblotting analysis as indicated. d A total of 8 × 106 THP-1 stable transfectants (which stably express Flag-RKIP) were differentiated with 100 nM PMA overnight in 6 cm dishes, primed with LPS and treated with nigericin (Ni), flagellin and poly(dA:dT). Whole cell lysates were immunoprecipitated with anti-ASC antibody or control IgG, followed by immunoblotting analysis as indicated. e GST-tagged fusion proteins (GST-RKIP or GST-null) were mixed with Flag-ASC, and GST beads were added to pulldown buffer for the in vitro pulldown assay. Pulldown and input samples were immunoblotted with anti-GST or anti-Flag antibody. Data are representative of two independent experiments