FIG 1.

Linearly ubiquitinated NEMO activates the IKK complex more efficiently than unanchored linear ubiquitin chains. (A) IKK complex (0.5, 2.5, or 5 μg/ml) containing either WT or R316A/R319A/E320A NEMO was incubated for 1 h at 30°C with GST-IκBα (amino acids 1 to 54), E1, and UbcH5c in the presence or absence of LUBAC, and the reaction mixtures were probed with the indicated antibodies. (B) Reaction mixtures containing E1, E2, LUBAC, and ubiquitin were incubated with or without ATP. After incubation, E1, E2, and LUBAC were inactivated with EDTA and NEM, and the reaction mixtures were dialyzed. The dialyzed samples were incubated with GST-IκBα (amino acids 1 to 54) and the IKK complex in the presence or absence of E1, E2, and LUBAC, followed by probing with the indicated antibodies. (C) His-HA-Ub₂ (10, 50, or 250 μg/ml) or 250 μg/ml of ubiquitin was incubated with E1, UbcH5c, LUBAC, IKK complex, and GST-IκBα (amino acids 1 to 54), followed by probing with the indicated antibodies.