(see insert Fig. 1A, lower panel). In Figure 1B, upper panel, samples obtained from separation of Raji cell lysates were mixed with reducing SDS sample buffer, boiled, separated by SDS PAGE and immunoblotted for DRa. The gradient was repro- duced in more than ten experiments, showing no more variation than one fraction. Chemical crosslinking To 100 ml of a pool of fractions 7 and 8 or of fractions 14 and 15 of the sucrose gradient (without Tris) separated Raji cell lysates increasing amounts of the thiol- cleavable crosslinker 3,39-dithiobis [succinimidyl propionate] (DSP) were added. Samples were incubated for 2 h on ice. For quenching the excess crosslinker, 10 ml of 1 M Tris was added. Samples were incubated with non- reducing sample buffer, boiled for 5 min, and subjected to SDS- PAGE. Supporting Information Figure S1 Carbohydrate processing, peptide binding of transgenic MHCII molecules and stability of MHCII-Ii complexes. A) MelJuSo cells were stably transfected with a V5- tagged DPb encoding cDNA. The lysate of the DPb–transfected MelJuSo cell clone was treated with PNGase F (lane 2), or with EndoH (lane 3). The digested (lane 2 and 3) and non-digested (lane 1) lysates were immunoblotted for DPbV5 chain. B) Intracellular transport of DRbV5 was monitored by digestion of cell lysate with PNGase F (lane2), or with EndoH (lane 3). Non-digested cell lysate was separated in lane 1. Lysates from non-transfected cells were analyzed in lanes 4 to 6. SDS-PAGE separated lysates were blotted for DRbV5 (lanes 1 to 3) and for DRb (lanes 4 to 6). C) Cell lysate from DRbV5-transfected MelJuSo cells was incubated for 1 h in SDS sample buffer at RT (lane 1). Lane 2 shows cell lysate boiled for 5 min in SDS buffer. The SDS separated lysates were immunoblotted for DRbV5. The position of the peptide-bound DRabV5 heterodimer and of the dissociated DRbV5 chain is indicated. D) MelJuSo (MJ) and MJDRbV5 cells were stained with CD63-Alexa 488 and with V5 monoclonal antibodies and examined by confocal-immunofluorescence microscopy. The left panels show CD63 and the middle panels V5 staining. In the right panel the two patterns were merged. E) MJDRaV5 cells were lysed and immunoprecipitated with V5 mAb. The immunopre- cipitate (lane 1) and the lysate (lane 2) were separated by SDS PAGE and western blotted with mAb Bu43 for Ii (notice that cell lysate contains DR-associated Ii and an excess of free Ii). The References 1. Marks MS, Blum JS, Cresswell P (1990) Invariant Chain Trimers Are Sequestered in the Rough Endoplasmic Reticulum in the Absence of Association with HLA Class II Antigens. J Cell Biol 111: 839–855. 2. Park S-J, Sadegh-Nasser S, Wiley DC (1995) Invariant chain made in Escherichia coli has an exposed N-terminal segment that blocks antigen binding to HLA- DR1 and a trimeric C-terminal segment that binds empty HLA-DR1. Proc Natl Acad Sci USA 92: 11289–11293. 3. Jasanoff A, Wagner G, Wiley DC (1998) Structure of a trimeric domain of the MHC class II-associated chaperonin and targeting protein Ii. EMBO J 17: 6812–6818. 4. Romagnoli P, Germain RN (1994) The CLIP region of invariant chain plays a critical role in regulating major histocompatibility complex class II folding, transport, and peptide occupancy. J Exp Med 180: 1107–1113. 5. Avva RR, Cresswell P (1994) In Vivo and In Vitro Formation and Dissociation of HLA-DR Complexes with Invariant Chain-Derived Peptides. Immunity 1: 763–774. 6. Ghosh P, Amaya M, Mellins E, Wiley DC (1995) The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3. Nature 378: 457–462. 7. Siebenkotten IM, Carstens C, Koch N (1998) Identification of a sequence that mediates promiscuous binding of invariant chain to major histocompatibility complex class II allotypes. J Immunol 160: 3355–3362. HLA-Class II-Invariant Chain Complexes position of Ii and of H and L chains of mAb V5 are shown on the left. F) Raji cells were lysed in 1% digitonin and Ii was immunoprecipitated with mAbs Vic-Y1 and Bu45. Immunopre- cipitates were washed with digitonin buffer and incubated with 1% Triton X-100. The immunoprecipitate (lanes 1 and 3) and the Triton X-100 supernatant (lanes 2 and 4) were western blotted for Ii (lanes 1 and 2) (mAb Bu43) and for DRa (lanes 3 and 4) (mAb TAL-1B5). The positions of Ii, of DRa and of immunoglobulin H and L chains are indicated by arrows. (TIF) Figure S2 Isolation of DRbV5 from transfected MelJuSo cell lysates. A) Lysates from the DRbV5-transfected MelJuSo cell line were immunoprecipitated for DRbV5 (monoclonal antibody V5, lane 1), for DRbHis (monoclonal antibody His, lane 2), for the DRab heterodimer (monoclonal antibody I251SB, lane 3), and for DRa (monoclonal antibody TAL-1B5, lane 4). Lysates from DRbV5 and from untransfected MelJuSo cells were separated in lanes 5 and 6. Three parallel blots were immuno- stained for DRb (S35, upper panel), for DRbV5 (monoclonal antibodyV5, middle panel) and for DRa (monoclonal antibody TAL-1B5, lower panel). Arrows indicate the positions of DRb, of DRbV5 and of DRa. B) DRbV5 and untransfected MelJuSo cells (MJ) were lysed in 1% digitonin. DRab was immunoprecipitated by the conformation-dependent mAb ISCR3. Immunoprecipitates and cell lysates were SDS-PAGE separated and western blotted for DRb (left panel, mAb LGII-612.14), for DRa (middle panel, mAb TAL-1B5), or for Ii (right panel, mAb Bu43). The positions of DRbV5, DRb, DRa and of Ii are indicated by arrows. (TIF) Text S1 (DOC) Supplemental text. Acknowledgments We thank Drs P. Roche and P. Cresswell for comments to the manuscript and Dr. G. Moldenhauer for antibodies. Author Contributions Conceived and designed the experiments: NK MZ AK ST JN SS. Performed the experiments: AK ST. Analyzed the data: NK. Wrote the paper: NK SS. Performed biochemical experiments: NK AK ST. Constructed DNA plasmids: JN. Modelled MHCII-Ii structure: MZ. 8. Ashman JB, Miller J (1999) A role for the transmembrane domain in the trimerization of the MHC Class II-associates invariant chain. J Immunol 163: 2704–2712. 9. Castellino F, Han R, Germain RN (2001) The transmembrane segment of invariant chain mediates binding to MHC class II molecules in a CLIP- independent manner. Eur J Immunol 31: 841–850. 10. Neumann J, Koch N (2006) A novel domain on HLA-DRb chain regulates the chaperone role of invariant chain. J Cell Sci 119: 4207–4212. 11. King G, Dixon AM (2010) Evidence for role of transmembrane helix-helix interactions in the assembly of the class II major histocompatibility complex. Mol Biosystems 6: 1650–1661. 12. Roche PA, Marks MS, Cresswell P (1991) Formation of a nine-subunit complex by HLA class II glycoproteins and the invariant chain. Nature 354: 392–394. 13. Lamb CA, Cresswell P (1992) Assembly and transport properties of invariant chain trimers and HLA-DR invariant chain complexes. J Immunol 148: 3478–3482. 14. Cresswell P (1994) Assembly, Transport, and Function of MHC Class II Molecules. Ann Rev Immunol 12: 259–293. 15. Jones PP, Murphy DB, McDevitt HO (1978) Two-gene control of the expression of a murine Ia antigen. J Exp Med 148: 925–939. 16. Arunachalam B, Lamb CA, Cresswell P (1994) Transport properties of free and MHC class II-associates oligomers containing different isoforms of human invariant chain. Int Immunol 6: 439–451. PLoS ONE | www.plosone.org 12 February 2011 | Volume 6 | Issue 2 | e17257