HLA-Class II-Invariant Chain Complexes Figure 3. Detection of Ii in sucrose gradient fractions and delineation of DR and Ii complexes. A) SDS-PAGE separated Raji cell extracts from fractions 1 to 24 and western blotting for Ii (monoclonal antibody Bu43) (upper panel) results in two peaks (scan lower panel). B) Immunoprecipitation of DR, and SDS-PAGE separation was conducted as in Fig. 2C, followed by immunoblotting for Ii (monoclonal antibody Bu43, upper panel) and evaluation as a scan (lower panel). The DR-associated Ii appears in lanes 12 to 16. C) DR and Ii oligomers displayed in a sceme. Peptide-loaded DR (pDRab) largely co-migrates in the sucrose gradient with the Ii trimer. Complexes containing DR-Ii are separated in fractions 12 to 16. doi:10.1371/journal.pone.0017257.g003 on a sucrose gradient, and samples from the low and from the high molecular weight peaks of DR in Fig. 1B were treated with the chemical crosslinker, 3,39- dithiobis [succinimidyl propionate] (DSP) and separated by SDS-PAGE followed by immunoblotting for DRa or for Ii (Fig. 4A). At low concentrations of DSP, DRa from fractions 14/15, which contain the DR-Ii complex, remains monomeric, but at 100 and 200 mg/ml DSP, it shifts to a high molecular weight band that appears on top of the gel (Fig. 4A bottom left panel). The crosslinking of DR from fractions 7/8 (which correspond to peptide-bound DR molecules) with increasing concentration of DSP converts the DRa monomer into a band that has the expected size of the DRab dimer (55 kDa; Fig. 4A, top left panel). No other bands were detected suggesting that DR crosslinking in these fractions is specific and does not result in the crosslinking of DR to other cellular proteins. In striking contrast, blotting of the same crosslinked fractions for Ii yielded a smear of high molecular weight material at the top of the gel at 100 and 200 mg/ml of DSP, while monomeric, dimeric, and trimeric Ii appear as distinct bands (top and bottom, right panels). With increasing DSP concentra- tion, Ii3 (95 kDa) becomes abundant, and in fractions 14/15 (which contain the DR-Ii complex), high molecular weight material accumulates above the Ii trimer and, at 200 mg/ml DSP, appears as a compact band at the very top of the gel. Importantly, we noted that even in the absence of association with DR, in fractions 7/8 (upper right panel), high molecular weight crosslinks of Ii were formed. This demonstrates that Ii associates with cellular components other than MHCII, as described previously [25–30]. Since such additional Ii-interacting proteins may be contained in all Ii-complexes that are obtained by chemical crosslinking, it must be assumed that the crosslinked high molecular weight material that is detected by immunoblotting for DR and for Ii at the top of the gel (Fig. 4A, lower panels) does not solely consist of DR and Ii but contains other cellular proteins. To determine the size of the high molecular weight band that was obtained upon crosslinking of the DR-Ii complexes, we separated the crosslinked samples by ultracentrifugation on a sucrose gradient. We found that DRa bands did not correspond to a species of uniform size but were almost evenly distributed all across the high molecular weight area of the gradient (Fig. 4B). This demonstrates that chemical crosslinking produces DR complexes with larger size than obtained by separation of untreated cell lysates in a sucrose gradient (compare Fig. 4B to Fig. 1B). Presumably, unstable and transient interactions of Ii with cellular components are stabilized by the crosslinking reagent. This might have led previous workers to propose a larger apparent size of the DR-Ii complex. HLA- DR, DP, and DQ heterodimers segregate into individual complexes with Ii If Ii3 associates with more than one MHCII dimer, one would assume that such complexes contain a mixture of MHCII isotypes PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e17257