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Fig. 4. Transport and assembly of YWD-mutated DRß with the DR
chain. IMRS cells were transiently transfected with DR and DRß, or with DR and YWD-mutated DRß in the presence (lanes 4, 5, 6, 8 and 9) or absence (lanes 1, 2, 3 and 7) of Ii. Cells were lysed and DR was immunoprecipitated with mAb I251. Immunoprecipitates were digested with Endo H (EH) (lanes 2 and 5), with PNgase F (PF) (lanes 3 and 6) or left untreated (Ø) (lanes 1 and 4). Subsequently the samples were separated by SDS-PAGE and immunoblotted with S35 for DRß. Lanes 7 and 8 show cell lysates western blotted for DR. Ii was immunodetected in lane 9. (B) Endo H digestion of transfected DRß chains. MelJuso cells were transfected with V5-tagged wtDRß (lanes 1-3), or the mutants DRßY (lanes 4-6), DRßW (lanes 7-9), DRßD (lanes 10-12), DRßYW (lanes 13-15) and DRßYWD (lanes 16-17). Lysates were digested with EndoH or PNGaseF and analyzed by western blotting for the presence of the V5-tagged DRß chains. (C) Cellular distribution of DRß chains. MelJuso cells were transfected with V5-tagged DRßYWD, with wtDRß or with empty expression vector (mock). Expression was monitored by double immunofluorescence staining of the cells with mAb V5 or DR mAb I251 (red staining, top row) and with an antibody against Cath B (green staining, middle row). In the bottom row, staining of rows 1 and 2 were merged. Nuclei were counterstained with DAPI. (D) MelJuso cells expressing V5-tagged wtDRß or YWD-mutated DRß were surface biotinylated and lysed with NP40. Cell lysates with wtDRß or YWD-mutated DRß were separated in lanes 3 and 4 or subjected to immunoprecipitation with V5 mAb. DRß chains were immunoblotted with streptavidin peroxidase (lanes 1 and 2) or with V5 mAb. (E) Co-immunprecipitation of DRß with Ii. MelJuso cells were transfected with V5-tagged wtDRß or V5-tagged mutant DRßYWD. Ii was immunoprecipitated with polyclonal antibody S22 and the immunocomplexes were detected by western blotting with mAb V5 for the presence of wtDRß (lane 1) or DRßYWD (lane 3). Expression of the V5-tagged ß chains was examined by western detection of the lysates (lanes 2 and 4). (F) Western blotting of DR immunoisolated with YWD mutant DRß chain expressed in the presence or absence of wtDRß. IMRS cells were transfected with DR, DRßYWD, Ii and wtDRß (lane 2) or with a vector control (lane 1). DRßYWD was immunoprecipitated and the immunocomplexes were western blotted for the presence of DR. Expression of the individual molecules was examined by western blotting of the lysates with mAb 1B5 (DR; lanes 4 and 5), mAb V5 (DRßYWD; lanes 6 and 7) and mAb 6D4 (wtDRß; lane 8).
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