To assess which one of the two peaks in Fig. 1B contained peptide- bound DR molecules, fractions were treated with sodium dodecyl sulphate (SDS) at room temperature prior to SDS-PAGE, since DR heterodimers with a bound peptide resist SDS-induced dissociation under these conditions [22]. An SDS-resistant band at ca. 55 kDa, corresponding to peptide-loaded DRab complexes (pDRab), was indeed observed in fractions 7 to 9 (Fig. 2A). No pDRab band was detected beyond fraction 11, indicating that this range of the gradient contains no peptide-loaded DR heterodimers. This suggests that fractions 13 to 15 contain the complexes of peptide-free DR with Ii. To further examine the composition of the separated DR complexes, we next subjected the gradient fractions to immunoprecipitation with anti-DR monoclonal antibodies. The anti-DR monoclonal antibody 16.23, which reacts with DRab heterodimers but not with DR-Ii oligomers [23], only immunoprecipitates DR from fractions 7 to 9 (Fig. 2B). In contrast, the conformation-dependent monoclonal antibody ISCR3 immunoprecipitates DR from both peaks (Fig. 2C), demonstrating the presence of DRab dimers. We next probed SDS-PAGE separated fractions for Ii and again found two peaks, in fractions 8 to 11 and 13 to15 (Fig. 3A). In contrast, blotting of the DR immunoprecipitates for Ii (Fig. 3B) resulted in a single peak at fractions 13 to15, which therefore contains the DR-Ii oligomer. No signal for Ii was obtained in fractions 6 to 11, suggesting that in the low molecular weight range of the gradient, Ii is present but not associated with the DRab heterodimer. Immuno- precipitation of sucrose gradient separated DC lysates with a monoclonal antibody to DR and subsequent blotting for Ii showed HLA-Class II-Invariant Chain Complexes again that Ii was only detected in fractions 13 to 16 (data not shown), indicating that these fractions contain DR-Ii complexes. The results of the sucrose gradient experiments are summarized in Fig. 3C. The pDRab complex displays the lowest mobility of the examined complexes (fractions 7 to 9). The low molecular weight peak of the Ii western blot (Fig. 3A), is found in fractions 8 to 11, slightly heavier than the pDRab complex (Fig. 2A) and it may represent Ii3. The peaks of pDRab and Ii3 overlap to a great extent, since molecules of this size are not completely separated on this sucrose gradient. Crucially, however, even though DR and Ii are both present in this region of the gradient, no interaction between them can be detected. The high molecular weight peak of DR in Fig. 2C and the peak of the DR-Ii complex in Fig. 3B both most likely represent the DRabIi oligomer. This complex comigrates with immunoglobulin G (150 kDa), which suggests that is significantly smaller than the estimated molecular weight of the postulated nonameric complex (DRab)3(Ii)3, 260 kDa [12]. Remarkably, the amount of oligomeric DR-Ii is lower than the amount of DRab heterodimers. From the density scans in Fig. 1B and Fig. 2C, we estimate that under the steady state conditions of this experiment, roughly one third of the DR molecules in the cell are contained in DR-Ii complexes. Assessment of DR and Ii oligomers by chemical crosslinking To demonstrate association between DR and Ii in an independent experimental system, Raji cell lysates were separated Figure 2. SDS-PAGE separation of Raji cell extracts from sucrose gradient fractions. The lower panels depict densitometric scans of the western blots. A) Samples treated with SDS sample buffer at room temperature were SDS-PAGE separated and immunoblotted for DRa (monoclonal antibody TAL-1B5). The upper panel shows SDS resistant pDRab bands (ca. 55 kDa) in fractions 6 to 11. The Scan represents both the pDRab and the DRa bands. B) Immunoprecipitation (IP) of DRab with monoclonal antibody 16.23, and western blotting for DRa (upper panel). The monoclonal antibody 16.23 selectively immunoisolates DR from fraction 6 to 10 and shows no reactivity to DR in fractions 11 to 17. C) Immunoprecipitation from sucrose gradient fractions 6 to 17 with DR monoclonal antibody ISCR3. SDS-PAGE separation and western blotting for DRa (monoclonal antibody TAL-1B5) results in separation of two DR populations, which is visualized as two separate curves in the density scan. doi:10.1371/journal.pone.0017257.g002 PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e17257