FIGURE 3.

Endotoxin tolerization impairs LPS-induced IRAK1 interactions with TRAF6 and IKKγ and compromises LPS-induced K63-linked polyubiquitination of TRAF6 and IKKγ. After exposure to medium or 10 ng/ml LPS for 20 h, THP1 cells or monocytes were washed and treated with medium or challenged with 100 ng/ml LPS for the indicated time course. A and B, TRAF6 and IKKγ proteins were immunoprecipitated (IP) and subjected to Western blot analyses with α-IRAK1 Ab (A) or α-Ub Ab (B). C, whole cell lysates of human monocytes were immunoprecipitated with α-Ub Ab, and the proportion of TRAF6 was detected by immunoblot (IB) analysis using α-TRAF6 Ab. A–C, whole cell lysates were examined by immunoblotting, using Abs against IRAK1, TRAF6, IKKγ, and Ub to determine their total protein levels and with α-p-p38 Ab to control for LPS inducibility and tolerization. Immunoblotting of total p38 or tubulin was performed to control protein loading. The results of a representative experiment (n = 3) are shown.