Figure 1.

Gene, Product and Expression of Peptide/β₂m/CD3-ζ

(A) Genetic design. Selected restriction sites are shown. br, bridge; lead, leader peptide; li, linker; p, peptide; pr, promoter; tm+cyt, transmembrane and cytoplasmic domain. (B) Scheme of an intact, surface MHC-I complex comprising the chimeric peptide/β₂m/CD3-ζ chain. (C) Flow cytometric analysis for surface expression. Splenic CD8 T cells of BALB/c and NOD mice (upper panel) and B3Z cells (lower panel) were transfected with 10 μg mRNA encoding the indicated constructs. mRNA encoding H-2K^d was added to enable presentation of the respective peptides. Cells were stained with anti-human β₂m and detected with FITC-conjugated donkey anti-mouse IgG. Staining of non-transfected cells is presented by the gray histograms. (D) Flow cytometric analysis of B3Z cells electroporated with different amounts of mRNA (in micrograms), no mRNA, or EGFP mRNA and analyzed using anti-human β₂m mAb purified from the supernatant of hybridoma HB149. (E) Western blot analysis performed under reduced conditions with cell lysates of B3Z cells transfected with the same mRNA as in (D), with detection using rabbit anti-human β₂m polyclonal antibodies (Abs).