LPS-induced expression of MHC II and costimulatory molecules at the plasma membrane. (A) D1 cells were cultured in 96-well plates and incubated for 0, 1, 3, 6, or 48 h with LPS. The surface expression of MHC II, CLIP-associated MHC II (MHC II/CLIP), CD40, and B7.2 was analyzed by FACS®. (B) Cells were cultured as above for 0, 1, 2, 3, 4, 5, 6, and 7 h with LPS either in the presence or absence of cycloheximide. Surface expression of B7.2 and MHC II was analyzed by FACS®. (C) Cells were lysed after 48 h of culture in the presence or absence of LPS in SDS sample buffer. Samples were incubated at room temperature, 37, 60, or 100°C and analyzed by Western blotting for MHC II β-chain. The Western blot shows monomeric MHC II β-chain and SDS stable MHC II. Upon stimulation with LPS, the amount of SDS stable MHC II/peptide complexes at room temperature strongly increased on expense of SDS unstable MHC II molecules. In contrast, the amount of SDS stable complexes at 100°C decreased after maturation. (D) Immature D1 cells secreted ∼7% of their β-hexosaminidase in a linear fashion during 18 h. The release increased only slightly after 4 and 8 h of LPS treatment but was unchanged at 18 h.