Figure 1 .
Effect of tumour necrosis factor α (TNF-α) stimulation and selective proteasome blockade on steady state IκBα concentrations (A), NF-κB binding activity (B), and RelA (p65) subcellular localisation (C). (A) Cells were pretreated with MG-132 (5 µg/ml; left panel), ALLN (50 µg/ml; right panel), or medium alone for 30 minutes and then stimulated for 0 to 60 minutes with TNF-α (10 ng/ml). Total protein was extracted and 20 µg subjected to SDS/PAGE followed by immunoblotting of IκBα using the ECL technique as described in the Methods section. Note that the IκBα standard used as a control contains seven extra amino acids (IκB-tag) compared with the endogenous epithelial IκBα. (B) Cells were treated as described above or infected for 12 hours with the Ad5IκB virus and then stimulated for 30 minutes with TNF-α (10 ng/ml). Nuclear extracts (5 µg) were tested for κB binding activity by electrophoretic mobility shift assay. Antibody supershifting is indicated by arrowheads. (C) Cells were treated as described in (A) and RelA localisation was visualised using an anti-RelA antibody followed by a rhodamine conjugated detection antibody.