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. 2017 Nov 2;18(12):2160–2171. doi: 10.15252/embr.201744480

Figure 4. NLRP11 promotes TRAF6 degradation in a MAVS‐dependent manner.

Figure 4

  1. 293T cells were transfected with Myc‐NLRP11 and Flag‐TRAF3, Flag‐TRAF5, or Flag‐TRAF6 for 24 h, and cell lysates were subjected to immunoprecipitation with anti‐Flag beads, followed by immunoblot analysis with indicated antibodies.
  2. NLRP11 and TRAF6 interaction was analyzed by co‐immunoprecipitation assay in THP‐1 monocytes upon Sendai virus (SeV) (MOI = 1) infection for 20 h.
  3. Luciferase activity in TRAF6 knockout (KO) 293T cells (bottom) transfected with ISRE promoter reporter and pRL‐TK plasmid and increasing amount of NLRP11 followed by SeV (MOI = 0.1) infection for 20 h. Data are expressed as means ± SEM of three independent experiments, versus cells transfected with EV with the same treatment, Student's t‐test, ns: no significant.
  4. Immunoblot analysis of indicated proteins in NLRP11 overexpressing THP‐1 cells followed with SeV (MOI = 1) infection for indicated time points (top). Numbers between two blots indicate densitometry of TRAF6 relative to that of β‐actin. Three independent experiments were quantified (bottom). Data are expressed as means ± SD of three independent experiments (**P < 0.01 versus uninfected cells, Student's t‐test).
  5. 293T cells were transfected with empty vector or Myc‐NLRP11 for 12 h, infected with SeV (MOI = 1) for 12 h, and then subjected to MG132 treatment for 6 h before harvesting. The cell lysates were subjected to immunoprecipitation with anti‐TRAF6 antibody followed by immunoblot analysis with indicated antibodies.
  6. Wild‐type (WT) and MAVS KO 293T cells were transfected with empty vector or Myc‐NLRP11 for 24 h, and the cells were then infected with SeV (MOI = 0.1) for indicated time points. Cell lysates were subjected to immunoblot analysis with indicated antibodies (top). Numbers between two blots indicate densitometry of TRAF6 relative to that of β‐actin. Three independent experiments were quantified (bottom). Data are expressed as means ± SD of three independent experiments (*P < 0.05 versus uninfected cells with the same treatment, Student's t‐test).
  7. WT and MAVS KO 293T cells were transfected with Myc‐NLRP11 and Flag‐TRAF6 for 24 h, and the cell lysates were analyzed by immunoprecipitation assay.

Source data are available online for this figure.