Analysis of SF2/ASF expression during MELC differentiation using cells sorted by flow cytometry. (A) Flow cytometry of MELCs at 0, 2, 4, and 6 days after DMSO-induced differentiation. MELCs were double-stained for a PE-conjugated anti-TER119 monoclonal antibody (mAb) and an FITC-conjugated anti-CD71 mAb and analyzed by flow cytometry. Axes indicate relative logarithmic fluorescence units for PE (x-axis) and FITC (y-axis). (B) Regions defined by characteristic staining pattern of day-4–induced MELCs, including CD71^highTER119^low (CD71⁺), CD71^highTER119^high (CD71⁺/TER119⁺), and TER119^highCD71^low (TER119⁺). (C) CD71⁺ and TER119⁺ cells were sorted by fluorescence-activated cell sorter (FACS) from day-4–induced MELCs and stained with May-Grünwald Giemsa. (D) Temporal relationship between 4.1R exon 16 splicing and SF2/ASF expression in differentiating MELCs. CD71⁺ and TER119⁺ cells were sorted from day-4–induced MELCs. Twenty micrograms of total proteins from CD71⁺ or TER119⁺ cells were analyzed with anti-SF2/ASF antibody. Immunoblotting with anti–β-actin antibody served as a loading control. RT-PCR was used to analyze exon 16 expression from total RNA isolated from the same samples.