Figure 4. Polyubiquitin chains induce NEMO-A20 interaction.

(A) HeLa cells were stimulated with IL-1β for the indicated time, then immunoprecipitation was performed with a NEMO antibody. Immunoprecipitate was used to measure IKK activity as in Figure 1A, or for immunoblotting with an A20 antibody. (B) S100 was incubated with ATP, −/+ TRAF6, −/+ the IKK inhibitor TPCA-1 (10 μM), for 1 hr. A NEMO antibody was used for immunoprecipitation. Proteins in the S100 and immunoprecipitate were detected by immunoblotting with the indicated antibodies. (C) As in (B), following depletion of UBC13 and/or addition of vOTU or (D) A20 WT or F770A/G771A was added to S100. Experiment was His₆-UBC13. performed as in (B) but without TPCA-1. (E) shUb-Ub (WT) or shUb-Ub (K63R) cells were untreated or treated with 1 μg/ml tetracycline for 4 days. Cells were then treated with TPCA-1 (20 μM) for 3 hr to enhance NEMO-A20 association. Cells were stimulated with IL-1β for the indicated times, then a NEMO antibody was used for immunoprecipitation. Proteins in the S100 and immunoprecipitate were detected by immunoblotting with the indicated antibodies. (F) MEF stable cell lines were treated for the indicated times with TNFα. Cell lysates were used for immunoprecipitation with a NEMO antibody. Cell lysate and immunoprecipitate were immunoblotted with the indicated antibodies.