Fig. 5: The DOC domain of HECTD3 interacts with NACHT/LRR domain of NLRP3 and blocks NLRP3-NEK7 interaction.
From: HECTD3 inhibits NLRP3 inflammasome assembly and activation by blocking NLRP3-NEK7 interaction
A HECTD3 and GST-tagged NLRP3 were transfected into HEK293T cells. GST-tagged NLRP3 was pulled down and detected with the indicated antibodies; B GST-tagged NLRP3 and 3×Flag-tagged HECTD3 were transfected into HEK293T cells. Flag-tagged HECTD3 was immunoprecipitated and detected with the indicated antibodies; C BMDMs isolated from WT mice were treated with LPS plus ATP. Cell lysates were immunoprecipitated by anti-NLRP3 antibodies and detected with the indicated antibodies; D BMDMs isolated from WT mice were treated with LPS plus ATP. The colocalization of NLRP3 and HECTD3 was detected by immunofluorescence; E Schematic representation of NLRP3; F Schematic representation of HECTD3; G HECTD3 and GST-tagged NLRP3 or GST-tagged NLRP3 truncated mutants were transfected into HEK293T cells. GST-tagged NLRP3 or GST-tagged NLRP3 truncated mutants were pulled down and detected with the indicated antibodies; H 3×Flag-tagged NLRP3 and GST-tagged HECTD3 truncated mutants were transfected into HEK293T cells. GST-tagged truncated HECTD3 mutants were pulled down and immunoblotted with the indicated antibodies. I, J HEK293T cells were transfected with Flag-tagged NLRP3 and GST-HECTD3 or GST-HECTD3-DOC, and were treated with 2.5 mM ATP. NLRP3 oligomerization in cytosolic pellets were analyzed with SDD–AGE assays (I); The interactions of NLRP3-NEK7 were detected by immunoprecipitation and immunoblot assays (J). K HEK293T cells were transfected with Flag-tagged NLRP3, Myc-tagged ASC and GST-HECTD3 or GST-HECTD3-DOC. The interaction of NLRP3-ASC was detected by immunoprecipitation and immunoblot analysis (K). L, M HEK293T cells were transfected with Flag-NLRP3, Myc-ASC, Flag-pro-caspase-1, Flag-pro-IL-1β plasmids as well as GST-HECTD3 or GST-HECTD3-DOC, and were stimulated with 2.5 mM ATP. Supernatants (SN) and cell extracts lysate (Lys) were analyzed using immunoblot assay (L); Supernatant IL-1β were analyzed using ELISA, n = 3 per group (M). Data are the mean ± SEM, ns (non-significant), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001, two-tailed unpaired Student’s t test was used.
