SIRPγ and SIRPα expression in T cells. (A) FACS analysis of resting human CD3⁺ T cells and PMNs stained with anti-SIRPα, anti-SIRPγ, or anti-MHC class I mAbs (solid histograms). The black solid lines represent a nonbinding isotype control mAb. (B) Equal amounts of total protein from lysates of resting PMNs and T cells were separated by SDS-PAGE under reducing conditions and analyzed by Western blotting for SIRPα expression (top). The membrane was stripped and reprobed with a SIRPγ mAb. The positions of SIRPα and SIRPγ proteins are marked. In addition, PMNs or T-cell lysates were loaded under reducing (lanes 1 and 3 from left) or nonreducing conditions (lanes 2 and 4 from left), electrophoresed, transferred to nitrocellulose membranes, and probed with a rabbit polyclonal Ab, which specifically recognizes the cytoplasmic tail of SIRPα (bottom). (C) FACS analysis of resting (black solid line) and 4 hours of TNF-α (black dotted line) treated HUVECs stained with anti-SIRPα (SE7C2; left), anti-SIRPγ (LSB2.20; right), or an isotype IgG control mAb (gray solid lines). These data are representative of 3 different preparations of cells.