FIG 3 .

PKR is inducibly ubiquitinated in tolerized macrophages, and tolerance depends upon a functional proteosome. (A) RAW 264.7 cells tolerized overnight with LPS (10 ng/ml) were pretreated for 30 min with either vehicle only (DMSO) or MG132 (25 µM). Cells were subsequently stimulated with LPS (250 ng/ml) for the indicated times. Whole-cell lysates were subjected to Western analysis and probed with the indicated antibodies. (B and C) Naive (B) or LPS-tolerized (C) RAW 264.7 cells were stimulated with LPS (250 ng/ml), and cells were lysed at the indicated times. PKR was immunoprecipitated from cell lysates, and immunoprecipitating complexes were subjected to SDS-PAGE. Separated complexes were sequentially probed with a monoclonal antibody directed against ubiquitin and a monoclonal antibody against PKR. Data are representative of 3 independent experiments. IB, immunoblotting.