MPL P106L cellular localization in transduced human MKs. (A) Retroviral construction. Cells were transduced with the retroviral vector pMEGIX-IRES-GFP containing either the human MPL WT or the mutated P106L tagged with HA. (B) Receptor expression on transduced human MKs: human MKs cultured 4 days with THPO were labeled with anti-HA and anti-CD41a antibodies and analyzed by flow cytometry 96 hours after transduction. MPL P106L is less expressed at the cell-surface membrane than the WT receptor, especially in mature MKs. A range from 3 × 10³ to 4 × 10⁴ cells were analyzed. (C) Human MPL localization: human CD34⁺ cells were transduced with the human MPL WT or P106L on the fourth day of culture with THPO and SCF. Four days later, cells were stained with either an anti-HA antibody to analyze MPL or an anti-CALR antibody to analyze the ER compartment. (D) Murine MPL-GFP localization: human CD34⁺ or murine lin⁻ cells were transduced with the pREX-IRES-CD4-muMPL-HA fused to GFP and analyzed by confocal microscopy at days 8 and 5, respectively.