Figure 5. REGγ interacts with IκBɛ and promotes its degradation.

(a) Expression of IκBɛ in colon epithelial cells isolated from WT and REGγ^−/− mice following 7 days of DSS treatment. Representative of four repeats (left). Densitometric analysis of IκBɛ relative to actin protein. Data represent means±s.e.m.; n=10 per group; ***P<0.001 (right). (b) HCT116 REGγ shR or shN control cells were treated with cycloheximide (100 μg ml⁻¹) for indicated times followed by western blotting. Representative images are from three repeats (left). Densitometric analysis of relative IκBɛ degradation. Data represent means±s.e.m.; *P<0.05, **P<0.01(right). (c) Immunoblot for HA-tagged IκBɛ with or without REGγ expressed from retroviral transgenes in NFκB-deficient cells. Representative images are from three repeats. (d) REG γ mediates proteolytic degradation of IκBɛ in a cell-free system. Purified REG γ, 20S proteasome, and in vitro-translated IκBɛ, IκBα and IκBβ were incubated as indicated and described in Methods followed by western blot analysis. Representative images are from three repeats. (e) IκBɛ accumulates in nucleus in REG γ-deficient colon epithelial cells. Colon epithelial cells collected from WT or REGγ^−/− mice at 0, 7 days post DSS administration were examined for IκBɛ after cytoplasm and nucleus separation by western blot analysis. Representative results were from two repeats. (f) Interactions between REGγ and IκBs. Pulldown assays were performed with 293T cells lysis transiently transfected flag- IκBɛ, IκBα or IκBβ and GST-REGγ. Representative of three repeats. (g) Interaction specificity between REGγ and IκBɛ. Purified REGγ and IκBs or IκBɛΔN60 were used in GST-pulldown analysis as described in Methods. Representative of four repeats. (h) Interactions between REGγ and IκBɛ in murine colon epithelial cell after 3 days DSS administration. Proteins were IP with an anti-REGγ Ab. Representative images were from three repeats. (i) Colon epithelial cells collected from WT and REGγ^−/− mice with or without prior exposure to DSS were IP with IκBɛ antibody and immunoblotted as indicated. Representative of two repeats.