Figure 2.

Accumulation of CD1b but not CD1c in lysosomal compartments and MIICs. Immature DCs were labeled for 22 h with radioactive [³⁵S]methionine/cysteine, homogenized, and subsequently fractionated by Percoll gradient centrifugation. (A) The relative distribution of the enzymes alkaline phosphodiesterase (APDE, ▵) and β-hexosaminidase (B-HEX, •) was determined to monitor the presence of plasma membrane or lysosomes in the Percoll gradient fractions, respectively. The distribution of total proteins (▪) is also shown. Membranes of each fraction were isolated and deglycosylated with PNGase F before immunoblotting with antibodies specific for TfR, Lamp-1, MHC class I, and CD1b and detection by chemiluminescence and autoradiography. (B) The distribution of MHC class II, MHC class I, CD1b, and CD1c molecules in Percoll fractions was compared by immunoprecipitation. Proteins from the postnuclear supernatant (PNS) were immunoprecipitated with either the specific antibody (Sp) or an isotype control antibody (Co). Only the CD1b and CD1c molecules were deglycosylated with PNGase F before SDS-PAGE.