Fig 5.

ESF prevents IκBα ubiquitination. (A) HCT-8 cells were pretreated with ETEC supernatant and then stimulated with TNF (20 ng/ml) for 30 min. Cell lysates were immunoprecipitated using an IκBα antibody and immunoblotted for both ubiquitin (Ub) and IκBα. Where indicated, HCT-8 cells were first treated with MG132 (50 μg/ml). (B) Quantification of ubiquitinated IκBα from immunoprecipitated samples (mean ± SEM; n = 3). Ubiquitinated IκBα intensity was normalized to immunoprecipitated IκBα abundance. Asterisks indicate IκBα abundance significantly different from that in the TNF-only lane. (C) IκBα immunoblotting after pretreating SW480 with 50 μM cycloheximide (CHX) for the indicated times. Where indicated, cells were first incubated with ETEC supernatant (2 h) before the addition of CHX.