FIGURE 4.

Induction of endotoxin tolerance inhibits K63-linked, but not K48-linked, polyubiquitination of TRAF6. THP1 cells were pretreated for 20 h with medium or 10 ng/ml LPS, washed and treated with medium or 100 ng/ml LPS for the indicated times. TRAF6 proteins were immunoprecipitated (IP) from whole cell lysates with α-TRAF6 Ab and immunoblotted (IB) with Abs discriminating K48- versus K63-linked polyubiquitin chains. Whole cell lysates were analyzed by immunoblotting using α-TRAF6, α-Ub, α-p-p38, and α-tubulin Abs to determine total expression of unmodified TRAF6, ubiquitin, and tubulin and to assess phosphorylation status of p38. Shown are the results of a representative (n = 3) experiment.