Figure 5. Inhibition of TNFα induced NF-κB signaling by iNUB.

(a,b) Effects of iNUB on TNFα and IL-1β-induced NF-κB signaling. MEFs were treated with 20 μM and 40 μM iNUB or DMSO 6 h before stimulation with TNFα (a) or IL-1β (b). NF-κB signaling was investigated by determining IκBα phosphorylation and degradation in Western Blot and NF-κB activation by EMSA. (c,d) iNUB does not affect JNK activation. MEFs were treated with DMSO or iNUB (40 μM) 6 h prior to stimulation with TNFα (c) or IL-1β (d). Phosphorylation of JNK and total JNK were analyzed by Western Blotting. (e,f) iNUB inhibits NF-κB target gene expression after TNFα, but not IL-1β stimulation. MEFs were treated with 20 μM and 40 μM iNUB or DMSO and stimulated with TNFα (e) or IL-1β (f). mRNA was isolated and NF-κB target genes were investigated by qRT-PCR. (n = 3; +/− SD) (g) iNUB induces apoptosis after TNFα stimulation. MEFs were stimulated for 24 h in the presence or absence of iNUB. Apoptosis was analyzed by AnnexinV staining and FACS. (n = 3; +/− SD) (h) iNUB impairs TNFα-induced IKK activation. MEFs were treated with 20 μM and 40 μM iNUB before TNFα stimulation. IKK activity after NEMO IP was determined by in vitro kinase assay using GST-IκBα 1–72 as the substrate. (i,j) iNUB does not directly inhibit IKK activity. The cellular IKK complex after TNFα stimulation and following NEMO IP (i) or recombinant IKKβ (j) were treated with iNUB before in vitro kinase reaction. IKKβ inhibitor (SC-514) served as positive control. (k) TNFα dependent recruitment of NEMO to ubiquitinated RIP1 via UBAN. ST-PD of StrepTagII-NEMO WT and D311N expressing MEF. Co-precipitation of ubiquitinated RIP1 in unstimulated or TNFα stimulated cells was performed and analyzed by Western Blot. (l) iNUB impairs recruitment of NEMO to ubiquitinated RIP1. ST-PD was performed with StrepTagII-NEMO WT after TNFα stimulation in the presence and absence of iNUB. Co-precipitated ubiquitinated RIP1 was analyzed by Western Blot.