Intact and cleaved forms of soluble uPAR increase in sera of G-CSF–treated donors. Cleaved forms of soluble uPAR are also released by donor PBMNCs in vitro. (A) ELISA measurement of soluble uPAR (suPAR) in the sera of 15 donors, collected before (day 0) or at different time points (days 3-5) of G-CSF administration. Error bars are not shown when graphically too small. (B) Immunoprecipitation and immunoblotting of sera obtained from 5 donors before (day 0) and at different time points (days 3-5) of G-CSF administration. Samples were immunoprecipitated with biotinylated monoclonal antibodies (mAbs R2 and R3), previously immobilized on streptavidin-coated beads, to detect intact (suPAR) and cleaved (c-suPAR) soluble forms of uPAR. Adsorbed proteins were deglycosylated by peptide-N-glycosidase, eluted, and analyzed by immunoblotting with a rabbit anti-uPAR polyclonal antibody. (C) Western blot analysis with an anti-uPAR polyclonal antibody of conditioned media obtained after serum-free short-term cultures of PBMNCs collected before (day 0) or at different time points (days 3-5) of G-CSF stimulation from 4 representative donors. The first and the last lanes of the panel were loaded with a positive control of intact soluble uPAR (suPAR) and a cleaved form of suPAR, devoid of D1 (c-suPAR), respectively.