FIGURE 2.

Endotoxin-tolerant THP1 cells exhibit impaired LPS-mediated K63-linked polyubiquitination of IRAK1. THP1 cells were pretreated for 20 h with medium or 10 ng/ml LPS, washed, and incubated with medium or 100 ng/ml LPS as indicated. IRAK1 was immunoprecipitated (IP) from cell lysates, followed by immunoblot (IB) analyses with anti-total Ub Ab or Abs specific for K63-linked Ub and for K48-linked Ub chains. For controlling total IRAK1 levels and determining cell activation and endotoxin tolerance induction, IRAK-1 and p38 phosphorylation were examined by immunoblotting of whole cell lysates with the corresponding Abs. Tubulin immunoblot was carried out to control protein loading. The data of a representative experiment (n = 3) are shown.