Samples Selleck Ulixertinib were analysed on 8% SDS–PAGE gels, transferred to nitrocellulose (BA85, Whatman), and probed with antibodies in PBS with 0·1% Tween-20 (PBST). Detection was performed by chemiluminescence with Femto Western reagents (Perbio, Cramlington, UK) and imaged on a Fuji LAS-3000
analyser. Densitometric analysis was performed using ImageJ (http://rsbweb.nih.gov/ij/). MHC class I molecules can be detected in a dimeric form on exosomes secreted from a number of different cell lines and in human plasma.15 The formation of these dimeric (molecular weights approximately 80 000–85 000) MHC class I structures, in the case of HLA-B27, is strictly dependent on the cysteine located at position 325 in the cytoplasmic tail domain, as demonstrated by immunoblotting of
exosomes secreted from the HLA-B27 transfected .221 human B-cell line expressing single amino acid substitutions of position 308 (C308A, cysteine to alanine) and position 325 (C325A, cysteine to alanine) in the HLA-B27 heavy chain, as shown in Fig. 1 (left panel). Removal of the cytoplasmic tail domain from the HLA-A2 molecule, which includes the unpaired cysteine at position 339, also prevents dimers check details forming in exosomes released from transfected rat C58 cells (Fig. 1, right panel). Hence cytoplasmic tail domain cysteine residues are crucial to the formation of exosomal MHC class I dimers. We identified a low level of glutathione in exosomes compared with whole cell lysates, which we proposed allowed the formation of these exosomal MHC Inositol monophosphatase 1 class I dimers by disulphide
linkages between unpaired cysteines in the tail domains. We also reported that treatment of cells with the strong oxidant diamide, which rapidly depletes intracellular glutathione, induced similar MHC class I dimers in the HLA-B27-expressing Jesthom B-cell line.15 To determine if the MHC class I dimers induced on whole cells by diamide were also controlled by the same tail domain cysteine, we treated HLA-B27-transfected CEM cells with diamide (Fig. 2a). Immunoblotting revealed the formation of HLA-B27 dimers in wild-type B27, and mutant C308A (cysteine 308 mutated to alanine). No dimers were induced in mutant C325A, demonstrating that cellular, oxidizing-induced MHC class I dimers are controlled by the same cysteines as in exosomes. Similar results were obtained with .221 cells transfected with the same B27 mutants (data not shown). Jesthom cells also displayed diamide-induced dimers, as previously reported (Fig. 2a). We also studied an HLA-B27 mutant (S42C) mutated to mimic the non-classical MHC class I molecule HLA-G, which forms extracellular dimers though cysteine at position 42. The HLA-B27.S42C mutant formed an enhanced level of dimer formation even in the absence of diamide, suggesting that it forms a similar structure to HLA-G. Diamide treatment failed to induce further dimer formation.