Disruption of ITAM signaling in DCs leads to the accumulation of ubiquitinated MHCII species and impaired recycling. (A) DCs were lysed, and MHCII was immunoprecipitated (clone M5/114), resolved by PAGE, and Western blots for ubiquitin (clone P4D1) and MHCII β chain (clone KL295) were performed. (B) The half-life of MHCII was determined in the presence and absence of chloroquine, to inhibit lysosomal proteolysis. DCs were pulse-labeled with [³⁵S] and chased for the indicated time points. Where indicated, chloroquine (50μM) was added 2 hours into the 9-hour chase. After chase, MHCII was immunoprecipitated, resolved by PAGE, and detected by autoradiography. (C) MHCII internalization was determined biochemically by using a surface biotinylating reagent that can be cleaved by MESNA, a cell impermeant reducing agent. DCs were first surface-biotinylated, chased for the indicated time points to allow internalization of MHCII, and treated with MESNA to remove biotin from MHCII that remained on the cell surface. Detection of biotinylated MHCII that had been internalized was achieved by immunoprecipitation with anti-MHCII antibody followed by Western blotting with streptavidin. Densitometric measurement of band intensity was used to calculate the percentage of internalized MHCII relative to total surface MHCII. Data are representative of 6 independent experiments. (D) Recycling of internalized MHCII back to the plasma membrane was determined biochemically. WT and DF DCs were surface biotinylated and chased for 20 minutes to allow internalization of recycling MHCII. Any biotinylated MHCII remaining on the surface of the cells was then removed by MESNA treatment. DCs were then chased for the indicated time points to allow internalized MHCII to reemerge on the plasma membrane, at which point biotin was removed with a second MESNA treatment. Detection of biotinylated MHCII that was trapped in intracellular compartments and protected from MESNA was achieved by immunoprecipitation with anti-MHCII antibody followed by Western blotting with streptavidin. During the course of the chase, loss of biotinylated MHCII indicates efficient recycling. Densitometric measurement of band intensity was used to calculate the relative amount of recycling MHCII that reemerged on the plasma membrane at each time point. Data are representative of 6 independent experiments; **P < .01.