LPS stimulates the translocation to the cell surface, but does not alter total cellular abundance of ENO-1 in human PBMos. (A) Flow cytometric analysis of human blood cells (R1; top left) for ENO-1 expression in PBMos identified as CD45/CD14-positive cells (R2; top right); histogram overlay illustrating ENO-1 cell-surface expression (open histogram) in PBMos and isotype control (shaded histogram; bottom left); histogram overlay of ENO-1 cell-surface expression (open histograms) on PBMos after stimulation with 5 μg/mL LPS for 6 hours (bold line) versus unstimulated (thin line) and isotype control (shaded histogram; bottom right). (B,C) Dose-response (left panels) and time course (right panels) of ENO-1 expression in PBMos after LPS stimulation as assessed by (B) real-time polymerase chain reaction (PCR) and (C) 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. (D) Western blot demonstrating ENO-1 cellular localization after stimulation of PBMos with 5 μg/mL LPS for 6 hours. Cells were surface-biotinylated and then lysed, and membrane proteins were separated from cytosolic fractions using streptavidin beads. The purity of cytosolic and cell membrane fractions was assessed by probing the samples for CD18 and P20S (the 20S subunit of the proteasome), respectively. The Western blot illustrated is from 1 representative experiment of 4. (E) Immunofluorescence for the detection of ENO-1 on unstimulated and LPS-treated (5 μg/mL, 6 hours) PBMos. Original magnification 63×/1.32-0.6 oil objective. Scale bar, 5 μm.