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Fig. 5. Detection of ectopic expression of either NACA (S-tagged-NACA) or EGFP proteins or transcripts in transduced CD34⁺ cells cultured under erythroid and granulocytic differentiation conditions. 72 hours after the initiation of the transduction with NACA or EGFP lentiviral vector, CD34⁺ progenitor cells were cultured for 10 days under conditions optimized for erythroid or granulocytic cell growth and maturation. Cells were then harvested and the presence of the proteins or transcripts was examined. (A) Ectopic expression of NACA or EGFP proteins. The proteins from cell lysates, either enriched on protein-S beads (for S-tagged-NACA protein detection) or not (for the EGFP protein detection) were detected by western-blot analysis with either an anti-NACA or an anti-EGFP immune serum. Lysates were tested for their total protein content using an antibody against the Grb2 adaptor protein. (B) Time-course analysis of EGFP expression during granulocytic differentiation of CD34⁺ infected cells. Cells were cultured for up to 10 days under conditions optimized for granulocytic growth and maturation. The EGFP expression was analysed at 3 days and 10 days of culture by flow cytometry. Positive EGFP fluorescence (gate M1) is set according to the untransduced control cells. `%' indicates the proportion of EGFP-positive cells. (C) PCR Detection of genomic integrations of the lentiviral TRIPU3-EF1-S-tagged-NACA vector in the granulocytic cells. PCR was performed on cells collected after 10 days of culture under conditions that encourage granulocytic growth and maturation using a sense primer in the EF1 promoter and an antisense primer at the 3' end of the NACA-encoding cDNA. (D) RT-PCR detection of the transcript encoding S-tagged-NACA in granulocytic cells. RT-PCR was performed on cDNA templates obtained from cells collected after 10 days of culture in conditions that encourage granulocytic growth and maturation using a sense primer in the S-tagged sequence and an antisense primer in the 3' end of the NACA sequence. (C,D) Products were electrophoresed on 1.2% agarose gels and visualized by ethidium-bromide staining. `Mix' represents the negative control without template. Gr, granulocytic cells.

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