Schematic diagram of wild-type ART2.2-GPI and ART2.2-Tm and stable expression of ART2.2-GPI and ART2.2-Tm by DC27.10 lymphoma cells. (A) Domain structures of GPI-anchored ART2.2 (ART-GPI) and transmembrane-anchored ART2.2 (ART2.2-Tm) are shown. The N-terminal signal peptide of ART2.2 was replaced by the signal peptide of CD8 (SP) followed by a FLAG-tag. The wild-type GPI signal sequence of ART2.2-GPI is underlined, and the predicted GPI anchor attachment site is indicated by the asterisk. In the ART2.2-Tm construct, the C-terminal GPI anchor signal sequence was replaced by the 21-residue transmembrane domain of CD8 (underlined) and 5 flanking extracellular and 17 cytosolic amino acid residues. (B) DC27.10 lymphoma cells were stably transfected with ART2.2-GPI or ART2.2-Tm. Stable transfectants were selected by growth in the presence of G418 and subcloned by limiting dilution. Solid lines indicate levels of ART2.2 expression on the surfaces of parental untransfected lymphoma cells and of ART2.2-transfectants, determined by FACS analysis after staining with Alexa488-conjugated ART2.2-specific mAb AliA53. Dashed lines indicate cells treated with Alexa488-conjugated isotype control mAb. Numbers indicate mean fluorescence intensity (MFI) of ART2.2 staining.