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. 2020 Apr 30;21(7):e49666. doi: 10.15252/embr.201949666

Figure EV5. EphA2 phosphorylates NLRP3 in vitro .

Figure EV5

  1. Immunoblot analysis of EphA2 and NLRP3 in AEC lysate; β‐actin served as a loading control throughout. Mock indicates unstimulated cells. 1 μg/ml polyI:C, 1 μg/ml dA:dT, and 500 ng/ml MSU were added for 10 h after 200 ng/ml LPS priming for 12 h. 5 mM ATP was added for 30 min. Reovirus was added for 10 h.
  2. Proteins were eluted and analyzed by immunoblot analysis with anti‐p‐Tyr antibody, anti‐HA antibody, and anti‐GFP antibody after in vitro kinase assay.
  3. Immunoblot analysis of NLRP3, IL‐1β, and cleaved IL‐1β p17 in lysates of the 16HBE cell line; β‐actin served as a loading control throughout. Cells were treated with a CRISPR vector control (sg‐Ctrl) or sgRNA targeting NLRP3 (sg‐NLRP3) with or without overexpression of Myc‐tagged NLRP3 or NLRP3 Y136. 500 ng/ml MSU was added for 3 h after 200 ng/ml LPS priming for 12 h.

Source data are available online for this figure.