FIGURE 5.

Deficient TLR4-induced TAK1 activation in LPS-tolerant cells. THP1 cells (A and B) and human monocytes (C) were pretreated for 20 h with medium or tolerized with 10 ng/ml LPS. The cells were washed and exposed to medium or 100 ng/ml LPS for the indicated time points (A and B) or for 15 min (C). TAK1 proteins were immunoprecipitated (IP) and analyzed by in vitro kinase assay (KA), using inactive MKK6 as a substrate (A and C, top blots), or subjected to immunoblot (IB) analyses with anti-p-TAK1 Ab (B). Whole cell lysates were analyzed by immunoblotting with Abs against p-TAK1 and TAK1 to determine TAK1 phosphorylation and total levels. Immunoblot analyses with Abs against IκB-α and p-p38 were performed to assess LPS activation/tolerance induction, and Abs against total p38 or tubulin were used to control for protein loading. Shown are the data of a representative (n = 3) experiment.