Figure 3

Induction of HLA-B27 heavy-chain dimers in KG-1 cells and human dendritic cells. KG-1, KG-1.B27sv5 and KG-1.B27.H114D.D116Ysv5 cells were stimulated with PMA and ionomycin for the indicated times (a) before preparation of cell lysates or surface biotinylation and (b) pulldown with streptavidin-agarose beads. Immunoblots were probed with HC10 or anti-tag pK. (c) In 24-hour stimulated KG-1 and KG-1.B27sv5, cell lysates were resolved under non-reducing conditions by two-dimensional gel electrophoresis, followed by immunoblotting with HC10 or pK. MHC class I dimers and monomers are indicated. (d) In human monocyte-derived dendritic cells were generated from peripheral blood monocytes of two HLA-B27-positive individuals and two negative controls, before being stimulated for 0, 24, 48 or 72 hours with lipopolysaccharides. Cells lysates were resolved under non-reducing conditions and immunoblotted for MHC-class I heavy chain (HC-10). Class I dimers and monomers are indicated. Molecular weight markers are shown in kDa.