Figure 4. MARCH5 interacts with the NACHT domain of NLRP3.

• A
  HEK293T cells were cotransfected with Myc‐MARCH5 and FLAG‐tagged NLRP3, ASC, or pro‐caspase‐1. Cell lysates were immunoprecipitated with anti‐FLAG‐M2 beads and detected with the indicated antibodies.
• B
  THP‐1 cells primed with 200 ng/ml LPS for 4 h were subsequently treated with or without 5 mM ATP for 30 min. Cell lysates were immunoprecipitated with IgG or anti‐NLRP3 antibody, and the indicated proteins were detected by immunoblotting.
• C
  March5 ^fl/fl BMDMs were treated with 200 ng/ml LPS for 4 h followed by 5 mM ATP for different durations. Cell lysates mixed with GST‐MARCH5 protein were pulled down with GST‐tagged beads, and the indicated proteins were detected by immunoblotting.
• D
  Schematic representation of NLRP3 wild‐type and truncated mutants (Upper). HEK293T cells were cotransfected with NLRP3 WT or NLRP3 mutants and SFB‐MARCH5 WT. Treatment with LPS (200 ng/ml for 4 h) and nigericin (15 μM for 60 min). Cell lysates were immunoprecipitated with streptavidin beads and immunoblotted with the indicated antibodies (Lower).

Source data are available online for this figure.