Fig 8.

LUBAC is essential for activation of the delayed phase of IKK. (A and B) RIP1 WT and KO MEFs were transfected with a scrambled control (Ctrl-A) or mouse HOIP-specific siRNA. Forty-eight hours after transfection, the cells were treated with TNF-α (10 ng/ml) as indicated, and IKK activation, p65 phosphorylation, and IκBα degradation were then examined by kinase assay and Western blotting. ns, nonspecific band. (C to E) RIP1 KO MEFs were transfected with a scrambled control or mouse HOIP-specific siRNA. Forty-eight hours after transfection, the cells were stimulated with TNF-α as indicated, and the ubiquitination of TRAF2 and cIAP1 in the total lysate (C), TRAF2 K63 ubiquitination within the TNFR1 complex (D), and recruitment of IKKβ, TAK1, TRAF2, and cIAP1 to TNFR1 (E) were then analyzed as in Fig. 4A and 5A and C.