HLA-Class II-Invariant Chain Complexes Figure 5. Inspection for co-isolation of DP and DQ isotypes with DR. A) MelJuSo cells were transiently transfected with cDNAs encoding V5- tagged DPa or DPb encoding cDNAs. Cells lysed in 1% NP40 were immunoprecipitated for DRab (monoclonal antibody ISCR3) (lanes 1,2,3,5,6 and 7). Lanes 4 and 8 contain cell lysates. The SDS-separated polypeptides were immunoblotted for DRa (lanes 1 and 5), for Ii (lanes 2 and 6), and for DPaV5 (lanes 3 and 4), or for DPbV5 chains (lanes 7 and 8). The position of DRa, Ii and Ab-epitope tagged DPaV5 or DPbV5 bands is indicated. H and L chain bands derived from the Ab used for immunoprecipitation are labeled on the left. B) Immunoprecipitates of the V5-tagged DPb (lanes 1 and 4) and of DRab (lanes 2 and 5) chains were separated by SDS-PAGE and either blotted for DPbV5 (lanes 1 to 3) or for DRa (lanes 4 and 5). To monitor expression of DPbV5, lane 3 contains cell lysate. The position of DPbV5, of DRa and of H and L Ig chains are indicated. C) MelJuSo cells were transfected with V5- tagged DQa cDNA. Cell lysate was immunoprecipitated for the V5-tagged DQa chains. Immunoprecipitates (lanes 4 to 6) and cell lysates (lanes 1 to 3) were digested with EndoH (EH), or with PNGase F (PF) or left untreated (Ø), separated by SDS-PAGE and western blotted as indicated. In lanes 7 to 9 MelJuSo cell lysates untreated and glycosidase treated were western blotted for DRa. doi:10.1371/journal.pone.0017257.g005 DRa were co-isolated in the immunoprecipitate, but that no DRa was detected in the Triton X-100 supernatant. This result indicates that Ii-DR complexes were stable in Triton X-100 and that in digitonin and Triton X-100 lysates, identical amounts of DR are associated with Ii. Thus, Triton X-100 induced dissociation is not an explanation for the lack of detection of nonameric Ii3(ab)3 complexes. According to the nonamer model, it should be possible to co- immunoprecipitate different MHCII isotypes that are bound to Ii in the nonameric complex directly with each other. To test this assertion, we next examined whether endogenous DR could be precipitated from the lysate of MelJuSo in a complex with stably expressed DPb tagged with the V5 epitope (DPbV5). DP or DR molecules were immunoprecipitated and immunoblotted for DPbV5 or for DRa, respectively. We found that immunoprecip- itation of DP and a subsequent western blot for V5 detects DPbV5 (Fig. 5B, lane 1), but not DRa (lane 4; correspondingly, the DR immunoprecipitate shows a band for DRa (lane 5), but not for DPbV5 (lane 2). These results suggest that transfected DPb and endogenous DR chains are not found in the same protein complex in cells. Next, we studied the co-isolation of transgenic DQa with endogenous DRa, hypothesizing that if more than one isotypic heterodimer is contained in a nonameric complex with Ii, it should be possible to co-isolate two isotypic a chains. We transfected MelJuSo cells with a cDNA encoding V5-tagged DQa and, to examine the functional assembly of this transgenic DQa with the endogenous b chain, we inspected the maturation of the DQa chain glycans. DQa chain contains two N-linked carbohydrates (upon arrival of the MHCII oligomer in the medial Golgi apparatus, one of the two glycans of the a chain acquires endoglycosidase H (EndoH) resistance, whereas the other remains EndoH sensitive). We found that most of DQa acquired partial EndoH resistance (Fig. 5C), which demonstrates that the transiently expressed DQa is transported out of the ER. To assess whether endogenous DRa co-isolates with the transfected DQaV5, DQaV5 was then immunoprecipitated with a monoclonal antibody against the V5 epitope. Western blotting of the immunoprecipitate showed a faint band of endogenous DRa (Fig. 5C, lane 4), which was completely sensitive to EndoH treatment (lane 5), indicating that these aggregates of DQ and DR were transient and did not leave the ER. It is important to emphasize that the amounts of DRa co-isolated with DQaV5 are traces of bands at the limit of detection. For comparison to lanes 1 to 3, glycosidase-treated lysates of MelJuSo cells were blotted for DRa (lanes 7 to 9). In conclusion, the results in Figure 5 indicate that the post-ER forms of DR, DQ and DP glycoproteins are not co-isolated from MelJuSo cell PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e17257