Fig. 4.

IκBα and IκBβ are constitutively degraded in most mutant cell lines whereas IKK is activated above basal levels only in mutant Z12. (A) Parental C6 and mutant cells were either untreated (-) or treated with cycloheximide (CHX) for 3 h. Cells were washed and lysed, and whole cell extracts were prepared. The amounts of protein in the extracts were quantitated, and equal amounts of the protein were analyzed by SDS/PAGE by using antibodies against IκBα. The same blot was probed with anti-IκBβ. The proteins were visualized by enhanced chemiluminescence. (B) IKK is activated above basal levels only in mutant Z12. Parental C6 and mutant cells were either untreated or treated with IL-1 for 15 min. The cells were washed and lysed, and the extracts were incubated with anti-IKKα antibody overnight at 4°C. The IKK complex was pulled down by using Protein A beads. The beads were washed, and a kinase assay was performed for 1 h in the presence of γ-labeled ATP and GST-tagged IκBα (residues 1–54) as a substrate. The beads were boiled in loading buffer, the proteins were separated by SDS/PAGE, and labeled IκBα was visualized by autoradiography.