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. 2020 Nov 12;21(12):e50421. doi: 10.15252/embr.202050421

Figure 5. Mechanism for PB1‐F2 interaction with NLRP3 hence preventing Nek7 binding.

Figure 5

  1. Schematic diagrams showing the NLRP3 constructs used in (B).
  2. 293T cells were co‐transfected with expression plasmids encoding for the indicated V5‐tagged domains of NLRP3 for 24 h. Whole cell lysate (left blot) and flag‐specific IP (right blot) were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of two independent experiments are shown.
  3. 293T cells were co‐transfected with expression plasmids encoding Flag‐NLRP3, V5‐PB1‐F2, and V5‐ASC for 24 h. Whole cell lysate and flag‐specific IP were analyzed by immunoblotting for indicated antigens. Equal loading of whole cell lysate was assured by blotting against beta actin. Representative blot of two independent repeats is shown.
  4. 293T cells were co‐transfected with expression plasmids encoding for Flag‐NLRP3, PB1‐F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and Flag IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown. Right panel: Quantification of IP bands’ intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‐tailed Student’s t‐test (*P < 0.05).
  5. As in (D): 293T cells were co‐transfected with expression plasmids encoding for Flag‐NLRP3‐LRR, PB1‐F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and NLRP3‐LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown.
  6. Quantification of IP bands’ intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‐tailed Student’s t‐test (*P < 0.05).
  7. As in (D and E): 293T cells were co‐transfected with expression plasmids encoding for Flag‐NLRP3‐LRR, PB1‐F2, or V5‐NLRP3‐Pyrin or combinations thereof as indicated. Whole cell lysate and NLRP3‐LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of three independent experiments are shown.

Source data are available online for this figure.