HLA-Class II-Invariant Chain Complexes from cell extracts, which suggests that these MHCII isotypes are not simultaneously contained in one complex with Ii. In a parallel approach, immunoprecipitation of DQ from separated MHCII-Ii complexes did not result in isolation of additional DR heterodimers as the nonamer model would suggest. To directly determine the size of DR-Ii complexes, we have estimated the size of the oligomer by ultracentrifugation on a sucrose gradient to be approximately 130 to 160 kDa. We thus present a pentamer model where only one single DR, DQ, or DP heterodimer is contained in a MHCII-Ii complex and propose a conformational change induced upon binding of one MHCII heterodimer to the Ii3 that could explain why class II molecules do not abundantly occupy all three CLIP sequences contained in the Ii3 complex. Results Size separation of DR and of Ii complexes In order to assess the molecular weight of cellular complexes of endogenous Ii and MHCII molecules, we prepared lysates from Raji cells, separated the solubilized proteins on a sucrose density gradient, and detected DRa by western blotting. The DRa band appeared in fractions 6 to 16 (Fig. 1B). A densitometric scan of the blot shows two peaks (Fig. 1B, lower panel) that correspond to molecular weights of 40–70 (fractions 7 to 9) and of 130–160 kDa (fractions 13 to 15), respectively, as determined by protein standards (Fig. 1A). The double band seen in fractions 12 to 16 of Fig. 1B is due to differential carbohydrate processing of the DRa glycoprotein, which was assessed by EndoH treatment of the DRa glycoprotein (data not shown). The lower band represents the endoplasmic reticulum (ER) form of the MHCII subunit, and the upper band contains DRa with complex type carbohydrates. In order to test whether separation of MHCII complexes is also achieved in primary cells, we used lysates of monocyte-derived dendritic cells (DC) for separation in a sucrose gradient. Fig. 1C shows a western blot of 24 fractions of sucrose gradient separated DC lysates. The scan of the DRa western blot reveals two peaks, similar to the result obtained with Raji cell extracts in Fig. 1B. Figure 1. Separation of cell extracts on a sucrose gradient by ultra-centrifugation. Cell lysates were ultra-centrifugated on a 0.4 to 0.8 M sucrose gradient at 100 000 g for 50 h. A) The sucrose concentrations of the fractions were determined after ultracentrifugation by a refractometer (upper panel). In a parallel tube, molecular weight standards were separated (lower panel). The SDS-PAGE separated protein standards were stained with Coomassie blue. The peak positions of the separated molecular weight markers are indicated by arrows. IgG bands (insert) are shown in fractions 14 and 15. B) Twenty four samples, obtained from the fractions of the sucrose gradient of Raji cell lysates were boiled in SDS sample buffer and analysed by SDS-PAGE and subsequent western blotting with monoclonal antibody TAL-1B5 against DRa (upper panel). C) Cell lysates from monocyte-derived human dendritic cells separated by the sucrose gradient were western blotted for DRa. The positions of DRa and of a protein standard (34 kDa) are indicated by arrows. The lower panels of B and C show densitometric scans of the western blots. doi:10.1371/journal.pone.0017257.g001 PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e17257