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. 2020 Jan 30;217(4):e20182091. doi: 10.1084/jem.20182091

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© 2020 Tang et al.

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Figure 4. — Cbl-b targets NLRP3 for K48-linked polyubiquitination. (A) Anti-His immunoblot analysis of Flag immunoprecipitates (IP) of lysates from HEK293T cells transfected with Flag-tagged NLRP3, His-tagged ubiquitin, and HA-tagged Cbl-b or Cbl-b C373A. CL, cell lysate; ctr, control. (B) Anti-ubiquitin immunoblot analysis of NLRP3 immunoprecipitates of lysates from WT and Cblb^C373A BMDMs primed with LPS (100 ng/ml, 4 h) and stimulated with ATP (2.5 mM, 5 min) and reprobed with anti–K48-ubiquitin and anti–K63-ubiquitin. (C) Anti-His immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3, HA-tagged Cbl-b together with His-tagged ubiquitin, His-tagged K48 ubiquitin, or His-tagged K63 ubiquitin. (D) NLRP3 immunoblot analysis of lysates from LPS-primed WT BMDMs pretreated with E-64 (10 µM) and MG-132 (5 µM) for 30 min and then stimulated with ATP or infected with EHEC for indicated time points. (E) NLRP3 and Cbl-b immunoblot analysis of lysates from WT and Cblb^C373A or Cblb^−/− BMDMs primed with 100 ng/ml LPS for 4 h and then stimulated with ATP or infected with EHEC for various time points. Data are representative of three independent experiments.