Fig. 4.

Extended nuclear localization and absence of IκBα are required for RelA ubiquitination. a IκBα inhibits RelA ubiquitination. HEK293T cells were transfected with his-ubiquitin, myc-RelA, and either empty vector (−) or HA-IκBα. Ubiquitination assay was performed under denaturing conditions. b Nuclear RelA S276A is not ubiquitinated in the presence of IκBα. HEK293T were transfected with myc-RelA S276A in the absence or presence of HA-IκBα. To maintain RelA nuclear with co-expression of IκBα LMB was added for 2 h. Assay was performed as in a. c Nuclear accumulation of RelA wt in the presence of IκBα does not lead to an increase of RelA ubiquitination. HEK293T cells transfected with myc-RelA and his-ubiquitin were treated with leptomycin B (LMB) for indicated time points. Cells were harvested in guanidine hydrochloride-containing buffer, and his pull-down was carried out. d Reduced nuclear translocation of RelA S276A results in diminished ubiquitination. HEK293T cells were transfected with his-ubiquitin and either empty vector control (−), RelA wt, RelA S276A or RelA S276A NLS mutant (mNLS). Ubiquitination of RelA was determined by his-ubiquitin pull-down and RelA antibody staining. IB immunoblot, PD pull-down