FIGURE 2.

NPC1^−/− DCs are activated normally by LPS and generate MHC-II-peptide complexes. A, immature or LPS-matured control (blue lines) or NPC1^−/− (red lines) DCs were monitored by flow cytometry for surface expression of total surface MHC-II using mAb M5/114. B, LPS-matured control or NPC1^−/− DCs were harvested and lysed in Triton X-100. Aliquots of the lysates were incubated with SDS-PAGE sample buffer and were either left at room temperature for 30 min or boiled for 3 min prior to SDS-PAGE. SDS stable pMHC-II complexes (53 kDa), total MHC-II α-chain (35 kDa), and actin (41 kDa) were detected by immunoblotting. The total amount of MHC-II present in each sample was quantitated by densitometry, normalized to the amount of actin present in the sample, and expressed as a percentage of total MHC-II present in control DCs.