lysates, and that therefore, the different isotypes most likely form entirely separate complexes with Ii. DRab heterodimers form pure isotype complexes with invariant chain The existence of such isotype-specific complexes of MHCII and Ii could be explained in two different ways. Either, an Ii homotrimer would form a complex with two or three MHCII heterodimers from one particular isotype only. Alternatively, one Ii homotrimer would only ever bind one MHCII heterodimer in total, of whatever isotype, to form a pentameric complex. To distinguish between these two possibilities, we next investigated whether the transgenic DRaV5 chain is found in the same complex with Ii and with endogenous DRa chain. We hypothesized that if this complex contains several DR heterodi- mers, it should be possible to detect two DRa chains in a complex with Ii. We therefore transfected MelJuSo cells with a V5-tagged DRa chain. Inspection of carbohydrate maturation showed that the majority of transfected DRa chains was partially EndoH resistant, indicating that they were assembled with endogenous b chains and transported beyond the cis-Golgi (Fig. 6A). To distinguish between endogenous DRa and transfected DRaV5, HLA-Class II-Invariant Chain Complexes we used the DRa-specific monoclonal antibody TAL-1B5, whose epitope is at the extreme C-terminus of DRa [31]; the extension of the C-terminus of DRa by the V5 epitope abrogates TAL-1B5 binding (Fig. 6B, lanes 1 and 3). When DRaV5 was immunopre- cipitated with the anti-V5 antibody and immunoprecipitates were blotted for co-isolated endogenous DRa with TAL-1B5, only very faint traces of endogenous DRa were detected (Fig. 6A, right panel). These very faint bands were entirely sensitive to EndoH treatment, suggesting weak and transient interactions between endogenous DRa and transfected DRaV5 in the ER only, but not at later stations of the intracellular pathway. To demonstrate separation between endogenous DRaand transfected DRaV5 in DR-Ii complexes by an independent experimental approach, we transiently expressed DRaV5, DRa, DRb, and Ii simultaneously in COS-7 cells (Fig. 6C). DRaV5 was immunoprecipitated from transfected and from untransfected COS cells (lanes 1 to 6) and immunoblotted for DRaV5, DRa and DRb. (As a control, the expression of the transfected cDNAs is shown in lanes 7 to 10). As before, no DRa was co- immunoprecipitated with DRaV5 (lane 4), while the DRaV5 chain (lane 2) and the DRb chain (lane 6) were readily detected in the V5 immunoprecipitates. Taken together, these experiments demonstrate that DRa and DRaV5 are contained in separate Figure 6. Isolation of Ab-epitope tagged DRa chains from transfected MelJuSo cells. A) MelJuSo cells transfected with DRaV5 cDNA were lysed. Cell lysates and V5-immunoprecipitates from cell lysates were digested with EndoH (EH) (lanes 2 and 5), with PNGse F (PF) (lanes 3 and 6), or left untreated (Ø) (lanes 1 and 4). SDS-PAGE separated cell lysates were immunoblotted for DRaV5 (monoclonal antibody V5) and V5-immunoprecipitated for DRa (western blotted with DRa monoclonal antibody TAL-1B5). B) Reactivity of monoclonal antibody TAL-1B5 to V5-tagged DRa is abolished. COS-7 cells were transfected with DRaV5 encoding cDNA. Cell lysate was immunoprecipitated with V5 or with TAL-1B5 (DRa) monoclonal antibodies. Immunoprecipitates were separated in lanes 1 to 4, and lane 5 contains lysate of MelJuSo cells. Lanes were western blotted as indicated. C) COS-7 cells were transfected with DRaV5, DRa, DRb and Ii encoding cDNAs. Cell lysates from transfected (+) and from non-transfected (2) COS cells were immunoprecipitated for V5 (lanes 1 to 6). Immunoprecipitates and cell lysates (lanes 7 to 10) were SDS-PAGE separated and western blotted with mAbs V5 for DRaV5, TAL-1B5 for DRa, LGII-612.14 for DRb, and Bu43 for Ii. doi:10.1371/journal.pone.0017257.g006 PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e17257