FIGURE 2.

Gain-of-function studies using HEK cells that express individual FUTs and/or PSGL-1. a, structure of pCS-CG vector containing FUT-EGFP fusion construct driven by human cytomegalovirus (CMV) promoter. b, HEK293T FUT4-EGFP (top row), FUT7-EGFP (middle row), and FUT9-EGFP (bottom row) cells viewed using differential interference contrast (DIC) (1st column), green channel due to EGFP in intracellular compartment (2nd column), red channel due to rabbit anti-human TGN-46 mAb and Alexa 597-conjugated secondary Ab, and merged image (Merge), which also includes blue DAPI nuclear stain. c, WT HEK293T (WT) and HEK-PSGL-1 (HP) cells were perfused over substrates bearing P-selectin-Fc, L-selectin-Fc, or E-selectin-Fc fusion proteins in a microfluidic flow cell. Anti-PSGL-1 mAb KPL-1 was added in some cases to HP cells to distinguish between PSGL-1-dependent and -independent cell adhesion components. Rolling (black bar) and adherent cell number (white bar) were measured. Wall shear stress was 1 dyne/cm² for all selectins. d–f, similar gain-of-function studies as in c were performed with HEK293T cells that overexpressed either FUT4-EGFP (d), FUT7-EGFP (e), or FUT9-EGFP (f). p < 0.05 with respect to WT alone (‡) or WT and HP+KPL-1 (†) for the same selectin interaction within the same figure panel. *, p < 0.05 with respect to the same selectin binding function measured with cells either lacking FUT (c) or having different FUTs (d–f).