HLA-Class II-Invariant Chain Complexes Figure 4. Chemical crosslinking of DR and of Ii oligomers. DR and Ii oligomers separated by sucrose gradient fractions of Raji cell lysates were crosslinked with DSP. A) Sucrose gradient fractions 7/8 and fractions 14/15 were analysed in the upper or lower panels. Concentration of DSP is shown on top. Untreated or crosslinked samples were separated by SDS-PAGE and western blotted for DRa (monoclonal antibody TAL-1B5, left panels) or for Ii (monoclonal antibody Bu43, right panels). On the right, positions of molecular weight markers are depicted. The dimeric form of Ii (70 kDa), which is seen both in untreated and in crosslinked samples, is produced by oxidation of a free cysteine residue of Ii to an inter-molecular disulfide bond [24]. B) Fractions 14/15 were crosslinked with 200 mg DSP/ml. Subsequently, crosslinked proteins were subjected to ultra- centrifugation on a sucrose gradient, separated by reducing SDS-PAGE followed by immunoblotting for DRa. doi:10.1371/journal.pone.0017257.g004 and that co-isolation of the Ii-bound forms of MHCII with each other should be possible. In contrast, if Ii3 and MHCIIab form pentameric complexes only, such co-isolation should not be possible. We therefore transiently transfected the MHCII- and Ii- expressing human melanoma cell line MelJuSo with cDNA encoding epitope-tagged DPa or DPb chains, lysed the cells and immunoprecipitated the endogenous DRab heterodimer. The immunoprecipitates and cell lysates were separated by SDS-PAGE and immunoblotted for the presence of transfected epitope-tagged DPa or DPb, or as a control for endogenous DRa and Ii. No co- isolation of DPa or DPb with DRa was detected, suggesting that different MHCII isotypes are not associated via Ii (Fig. 5A, lanes 3 and 7). In the next experiments, we again examined MHCII-Ii complexes for the presence of two different MHCII isotypes. To achieve a uniform expression of the transfected cDNA and to make sure that the transfected MHCII subunits are integrated into endogenous MHCII-Ii complexes and introduced into the biosynthetic route of MHCII molecules, we used stably transfected cell lines. We first demonstrated by carbohydrate maturation, endosomal localization, and formation of SDS-resistant dimers that the transgenic MHCII subunits are assembled and transport- ed together with endogenous MHCII (see Text S1, supporting information, Fig. S1A, B, C and D). Next, we tested the sensitivity of the co-isolation experiment. We immunoprecipitated the transfected V5-tagged MHCII chain and immunoblotted for co- isolated Ii. The result indicates that a substantial amount of Ii was co-isolated with the V5-tagged MHCII chain (Fig. S1E), and it suggests that any additional MHCII isotype heterodimers (which should be present in the same complex according to the nonamer model) should be readily detectable. Since this was not the case in the above experiment (Fig. 5A), we conclude that in our experimental system, nonameric Ii3(ab)3 complexes with mixed isotypes most likely do not exist. To make sure that a potential nonameric complex of MHCII and Ii had not been partially disrupted by the use of the detergent Triton X-100, we decided to test the stability of MHCII-Ii complexes in other detergents, especially the mild detergent digitonin. Ii was immunoprecipitated from a digitonin cell lysate and bound to Protein G Sepharose. This Ii-immunoprecipitate (which according to the nonamer model, should contain three MHCII heterodimers and Ii3) was then incubated with Triton X- 100 to test whether Triton X-100 would release any MHCII proteins. The immune complexes on the beads and the Triton X- 100 supernatant were then separated on SDS PAGE and immunoblotted for Ii and for DRa. Fig. S1F shows that Ii and PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e17257