FIG. 3.
F. tularensis infection supernatants induce ubiquitination of MHC class II. BMDM were pretreated or not pretreated with 100 U of IFN-γ/ml for 24 h and then treated with infection supernatant with or without 50 mM NH4Cl as described in the text. Ubiquitinated MHC class II from cell lysates was precipitated with P62-UBA-ubiquitin-binding agarose beads. Ubiquitinated and total MHC class II was analyzed by Western blotting. Exposure of BMDM to infection supernatant leads to increased ubiquitination of MHC class II, observable only with the addition of NH4Cl to prevent rapid class II degradation. Shown are representative results from one of two independent experiments.