NEMO and IRAK1 are ubiquitinated in IL-1–induced aggregates. U2OS cells left untreated or stimulated with IL-1 or TNF (for 12 and 5 min, respectively) were either directly lysed (TL) or permeabilized with saponin before lysis (After Sap.) as described in the Materials and methods. (right) NEMO immunoprecipitates from saponin-extracted cells were left untreated (−) or treated (+) with the vOTU deubiquitinase for 1 h at 37°C. Protein extracts were analyzed by immunoblotting for the detection of NEMO, IRAK1, and β-tubulin, as indicated.