Interaction of DAPK with NLRP3. (A) Interaction between DAPK and NLRP3 in HEK293T cells. The 293T cells were transfected with DAPK-Flag and/or NLRP3-Myc, as indicated, and cell lysates were prepared 48 hours later. Cell lysates were precipitated with anti-Flag or anti-Myc, and the amount of NLRP3-myc and DAPK-Flag in the precipitates determined by immunoblots. (B) Multiple domains of DAPK mediate interaction with NLRP3. Flag-tagged DAPK fragments containing kinase (K), calcium/calmodulin (CaM) regulatory domain, ankyrin repeats (AR), cytoskeleton binding (Cy), linking (Li), and death domain (DD), as indicated, were transfected into 293T cells together with NLRP3-Myc. Cell lysates were prepared 48 hours after transfection. The expression of each DAPK mutant in lysates (Input) was determined by immunoblots. DAPK mutant in lysates were pulled down by anti-Flag (M2) beads, and the associated NLRP3 detected by anti-Myc.