Fig. 6

DR is retained by DR_KKAA upon formation of nonameric-like structures. a Schematic representation of αSCD and DRβ and illustration of the rationale behind the use of αSCD with DRβ_KKAA. Left; DRα and Ii luminal domains are linked by a flexible gly₃/ser/gly₃ linker (αSCD). Top view shows the association of αSCD with a β chain. Middle; when co-expressed with DRβ_KKAA, DRβ will still egress the ER if αSCD does not trimerize via the TRIM domain. Right; formation of a trimer through TRIM will force the incorporation of both DRβ and DRβ_KKAA in the same complex, which will be ER retained. b HEK293T cells were mock-transfected or transfected with αSCD or with αSCD and DRβ. Cell lysates were treated with or without EndoH and blotted for DRα (DA6.147). Open arrowhead and arrowhead represent EndoH resistant forms of αSCD with different types of complex sugars. Star represents the EndoH sensitive αSCD. Arrow represents cleavage products of αSCD. Full-length blot is showed in Additional file 1: Figure 1F. c–e HEK293T cells were transiently transfected with DRβ_myc (DR) and/or DRβ_KKAA together with αSCD. After 48 h, cells were analyzed by flow cytometry to evaluate CLIP surface expression, using CerCLIP.1 (c), MHCII (d) and Ii (e) surface over total expression ratio using L243 and BU45, respectively. Representative histograms of CLIP surface expression (c), MHCII surface and total expression (d) and Ii surface and total expression (e) are shown. Ctrl represent isotype control antibodies. Error bars indicate the SD from at least three independent experiments. Student t-tests were performed; *p ≤ 0.001 and **p ≤ 0.05