LPS increases the cell-surface abundance of ENO-1 on U937 cells. (A) Histogram overlay of ENO-1 cell-surface expression (open histograms) on U937 cells after stimulation with 5 μg/mL LPS for 6 hours (bold line) versus unstimulated (thin line) and isotype control (shaded histogram). (B) Immunofluorescence for ENO-1 on unstimulated and LPS (5 μg/mL, 6 hours)–treated U937 cells. Original magnification 63×/1.32-0.6 oil objective. Bar size 5 μm. (C) Western blot demonstrating the cellular localization of ENO-1 after stimulation with 5 μg/mL LPS for 0 to 12 hours. Cells were surface-biotinylated, and streptavidin beads were used to separate membrane proteins from proteins resident in the cytosol. The purity of cell membrane and cytosolic fractions was assessed by probing the samples for P20S and CD18, respectively. The Western blot illustrated is from 1 representative experiment of 4. (D,E) Dose response (left panels) and time course (right panels) of ENO-1 expression in U937 cells after LPS stimulation as assessed by (D) real-time PCR and (E) Western blot. Real-time PCR results are expressed as the fold-increase in ENO-1 expression (normalized for β-actin expression) versus values obtained for unstimulated cells and are mean ± SD; n = 5. The Western blot illustrated is from 1 representative experiment of 4.