Selective down-regulation of surface MHCII endocytosis and destruction during DC maturation. (A) Freshly isolated (immature) or cultured (mature) splenic DCs were incubated on ice with biotinylated anti–MHC II mAb M5/114, washed, and then incubated at 37°C for 0 to 80 minutes. The amount of MHC II–mAb complexes remaining on the surface was then determined by staining with avidin–fluorescein isothiocyanate (avidin-FITC) and FACS analysis. The graph shows the values of the mean linear fluorescence (MLF) of each sample relative to the values at the 0 time point. The results are the average of 3 independent experiments. (B) Freshly isolated (immature) and cultured (mature) spleen DCs were cell-surface biotinylated at 4°C and then incubated for the times indicated at 37°C. Sequentially immunoprecipitated MHC II (top; the bands correspond to the MHC II α- and β-chains) and MHC I (middle; the band is the MHC I heavy chain) or total cell lysates (bottom) were run in 12.5% SDS-PAGE and analyzed by Western blot with the use of avidin-HRP. Since the cultured cells expressed several times more surface MHC I and II than their fresh counterparts, the exposure times of the pictures shown were adjusted so that the signals of the samples corresponding to the fresh and cultured cells at the 0 time points were comparable. The numbers at the left side of the panels show the relative amount of each indicated polypeptide that was lost between 0 and 80 minutes, as determined by gel densitometry. The results shown are representative of 3 independent experiments