Fig. 4.
RKIP inhibits the interaction between ASC and NLRP3 or NLRC4 to interfere with the inflammasome. a HA-ASC was cotransfected with Mock, Flag-NLRP1, Flag-NLRP3, Flag-NLRC4 and Myc-RKIP into 293T cells (4 × 105) as indicated, and cell lysates were immunoprecipitated with anti-Flag beads (M2 beads) after 48 h, followed by immunoblotting analysis as indicated. The star indicates the target bands. b RKIP expression was knocked down in 293T cells (2 × 105) by using a specific siRNA, and then, the cells were cotransfected with HA-ASC and Mock, Flag-NLRP1, Flag-NLRP3 or Flag-NLRC4. Cell lysates were immunoprecipitated with anti-Flag beads (M2 beads) after 48 h, and the samples were analyzed by immunoblotting as indicated. The star indicates the targeted bands. c, e A total of 8 × 106 WT or Rkip-KO PMs were primed with LPS and treated with nigericin (Ni) (c) or flagellin (e). Cell lysates were immunoprecipitated (IP) with anti-ASC antibody, followed by immunoblotting with the indicated antibodies. d, f Mock or THP-1 stable transfectants (8 × 106), which stably express Flag-RKIP, were differentiated with 100 nM PMA overnight in 6 cm dishes, primed with LPS and treated with nigericin (Ni) (d) or flagellin (f). Then, the cell lysates were immunoprecipitated (IP) with anti-ASC antibody, followed by immunoblotting with the indicated antibodies. Data are representative of three independent experiments