Fig. 3
Trimerization through TRIM is sufficient for the formation of high-order complexes. a Schematic representation of WT DRα + DRβ/Ii and DRα + βSCD. Top view highlights αβ interaction with the CLIP domain. Top view of an Ii3 with αβ dimers illustrates a nonamer (αβIi)3. In βSCD, Ii and DRβ luminal domains are linked by a flexible gly3/ser/gly3 linker. Top views show association of DRα to βSCD and trimerization of the complex via TRIM. b HEK293T were mock-transfected or transfected with DRα + DRβ + Ii or with DRα + βSCD. Cell lysates, with or without EndoH, were analysed by WB and blotted for DRβ (XD5 mAb). Arrowhead represents EndoH resistant WT DRβ. Open arrowhead and arrowhead represent βSCD and a cleavage product of βSCD, respectively, both resistant to EndoH. c DRα + βSCD were transiently transfected in HEK293T. After 48 h, cells were stained to detect MHCII, Ii and CLIP (L243, BU45 and CerCLIP.1 mAbs, respectively) (left histogram). Cells were transfected as above as well as with DM and stained for surface CLIP or permeabilized to detect DM (Map.DM1 mAb) (right histogram). d Cells were either mock-transfected, transfected with DRα + βSCD alone or with p33 or p35. Cell lysates (right lanes) and material precipitated with the Ii-specific Pin.1 mAb (left lanes) were analyzed by WB. DRα was detected using the DA6.147 mAb. H chains of Ig used for IPs are indicated. e Samples from (d) were analyzed using the XD5.117 mAb specific for the DRβ1 extracellular domain. In e, d, Ig identifies antibody heavy chain detected in lanes with IP products. f–h Cells were transfected with DRα + βSCD alone or with p35LIML or p35LIMLTRIM. f Cell surface MFI for CLIP (CerCLIP.1 mAb) are represented in bar charts. g, h MFI ratios obtained for surface versus total expression of MHCII (using L243) and Ii (using BU45) are represented in bar charts. Error bars indicate the SD from five independent experiments. Student t-tests were performed; *p ≤ 0.001 and **p ≤ 0.05. Full-length blots from this figure are presented in Additional file 1: Figure 1C–E