G-CSF–mobilized CD34⁺ HSCs do not express uPAR. (A) Immunophenotyping of CD34⁺ cells performed by 3-color flow cytometry on a mononuclear gate with anti-uPAR mAb, quantified as mean fluorescence intensity, in BM and in G-CSF–mobilized PB CD34⁺ HSCs from donors 1 to 3. (B) Western blot analysis with an anti-uPAR polyclonal antibody of BM CD34⁺ HSCs (BM HSC) and G-CSF–mobilized PB CD34⁺ HSCs (PB HSC) from donors 1 to 3 (100 μg total protein); PBMNCs from the same G-CSF–treated donors were used as positive controls (50 μg total protein). (C) RT-PCR analysis of uPAR mRNA expression in highly purified G-CSF–mobilized PB HSCs from a representative donor and THP-1 cells, used as a positive control. PCR in the presence of primers for GAPDH was used as a loading control.