Appendix figure 3. Analyzing p52 and p100 protein encoded by Nfkb2 in unstimulated cells and during signaling.
(A) Constitutive degradation of NF-κB bound (left panel) or free (right) IκBα (top panel) and p100/IκBδ (middle) were evaluated by immunoblotting respective extracts derived from WT (left) or NF-κB deficient (right) cells treated with 10 mg/ml of protein synthesis inhibitor cycloheximide. As compared to the stable NF-κB bound form, free IκBα degraded rapidly with a half-life ∼5 min. In contrast, both NF-κB bound p100/IκBδ present in WT cells (top) and free p100/IκBδ in NF-κB-deficient cells (bottom) were stable with a half-life > 12 hr. Actin (bottom panel) was used as loading control. The data represents two biological replicates. (B) Immunoblot revealing TNFR-induced IKK2 mediated degradation of NF-κB bound IκBα in WT (left panel, top) or free IκBα in NF-κB-deficient cells (right, top). Based on these data, IKK2-mediated degradation rates of the free and NF-κB bound classical IκBs were assigned comparable values in the model. Both in its bound (left, middle) or free (right, middle) form, p100/IκBδ were insensitive to IKK2 signals. (C) Immunoblot showing LTβR-induced NIK-IKK1 mediated degradation of NF-κB bound (top panel) and free (bottom) p100/IκBδ in WT and NF-κB deficient cells, respectively. Importantly, signal induced p52 accumulation was evident at 24 hr in WT, but not in NF-κB deficient, cells suggesting that newly generated p52 requires other NF-κB monomers for mutual stabilization. (D) Immunoblot of RelA co-immunoprecipitates revealing liberation of RelA from inhibitory p100/IκBδ (top panel) and generation of RelA:p52 complexes (middle) during LTβR signaling. Nfkb2−/− cell extracts were used as control (bottom).