FIG. 7.

Requirement of a functional cellular IKK complex for rapid LPS-induced proteolysis of p105. 70Z/3 cells (lanes 1 to 13), 1.3E2 cells (lanes 14 to 25), or 1.3E2 cells stably expressing HA-tagged IKKγ (lanes 26 to 29) were LPS stimulated for the indicated times in either the presence or absence of cycloheximide (CHX) or CHX and ALLN (30 min of incubation), as indicated. As a control, cells were treated with CHX or CHX and ALLN alone for 150 min (lanes 11, 12, 24, and 25). An EMSA with NF-κB DNA-binding activities is shown in the top panel. Migration of p50-p65 as well as of p50-p50 complexes is indicated. Detection of p105, p50, IκBα, IKKα, and IKKγ by Western blotting is shown below, and the migration of the specific bands is indicated. Please note that a nonspecific band migrates slightly slower than p105, as determined by peptide competition (lane 13).