Fig. 1.

Agonist-activated IL-36R is polyubiquitinated. a Polyubiquitination of IL-1Rrp2 after activation of IL-36R by the IL-36γ. NCI cells were not treated or treated with IL-36γ (10 ng/mL) and harvested over time. IL-1Rrp2 polyubiquitination was determined by immunoprecipitation of IL-1Rrp2 and Western blotting probed with an antibody to detect ubiquitin [12]. HC denotes the heavy chain of the immunoglobulin used in the immunoprecipitation assay. b Analysis of the ubiquitination linkages attached to IL-1Rrp2. NCI cells were activated with IL-36γ (10 ng/mL) and incubated for 6 h before the cell lysis with RIPA buffer. IL-1Rrp2 was immunoprecipitated by anti-IL-1Rrp2 antibody to treat with different deubiquitinases. USP2CD removes all ubiquitins, GST-AMSH removes K63-linked ubiquitins, OTUB1 moves K48-linked ubiquitins, and Cezanne removes K6 and K11 ubiquitins. Treated samples were then analyzed by Western blot probed with anti-ubiquitin antibody. The results shown are representative of 3 independent experiments.