Stimulus-dependent activation of IKK and IKKβ in human monocytic THP-1 cells. (A) Nonstimulated cells (lanes 1, 4), TNF-–stimulated (100 U/mL)(5 minutes) cells (lanes 2, 5), and LPSE coli 0127:B8 (1 μg/mL)-stimulated (30 minutes) cells (lanes 3, 6). Lanes 1-3 represent immunoprecipitates obtained with anti-IKK antibody; and lanes 4-6 represent immunoprecipitates obtained with anti IKKβ antibody. The IKK kinase assay was done as described in Materials and Methods by using IκB-GST fusion protein as the substrate. (B) IKK and IKKβ activity toward wild type IκB substrate and IκB mutant (S32A and S36A) substrate prepared as GST fusion proteins. The cells were stimulated with TNF- (100 U/mL) for 5 minutes and with LPS (1 μg/mL) for 30 minutes.