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Fig. 1 | BMC Immunology

Fig. 1

From: The transmembrane domain and luminal C-terminal region independently support invariant chain trimerization and assembly with MHCII into nonamers

Fig. 1

Formation of Ii trimers in absence of the TRIM domain. a Schematic representation of WT p35, p35LIML and p35LIMLTRIM. The glycosylation sites, the CLIP region, the TM and the RxR motif have been illustrated on the different p35 molecules. Leucine-based endosomal targeting motifs (di-leucine; open circles in p35) were mutated to alanines in p35LIML and p35LIMLTRIM. α-helices responsible for the C-terminal trimerization (TRIM domain) were deleted (Δ aa128 to 216 of p33) in the p35LIMLTRIM mutant. Loss of the BU45 mAb epitope is coped with by addition of a myc/His-tag (detection 9e10 mAb). The Ii top view highlights how the trimerization domains oligomerize. The top view of the IiTRIM mutant shows the myc tag and the trimerization via the TM domains (circles) of Ii moieties. b HEK293T cells were transiently transfected with empty (mock) plasmid, p35, p35LIML or p35LIMLTRIM. Cells were lysed in presence of reducible cross-linker DSP (400 µg/ml). Proteins were separated by SDS-PAGE in non-reducing conditions. Samples were blotted using a rabbit anti-CLIP serum. Monomeric (Ii1, TRIM1), dimeric (Ii2, TRIM2) and trimeric (Ii3, TRIM3) forms are indicated on the right. Full-length blot is presented in Additional file 1: Figure 1A. c Histograms depicts the densitometry of Western blot bands (trimers and dimers) in relation to bands associated with monomers for each molecule. The densitometry was performed on blots from 3 independent experiments, as shown in (b). d HEK293T cells were transiently transfected with an empty plasmid (mock) or plasmids expressing p35, p35LIMLTRIM or p35 and p35LIMLTRIM. Cells were lysed and immunoprecipitated using 9e10. Immunoprecipitated material and cells lysates were analyzed by SDS-PAGE and immunoblotted for WT p35 using Pin.1. Ig identifies antibody heavy chain detected in lanes with beads and IP products. Full-length blot is presented in Additional file 1: Figure 1B

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