Enrichment and characterization of N-Ras*–transduced erythroid cultures.

(A) Representative dot plots showing the 4-color immunophenotypic status of N-Ras*–transduced cultures: (left) CD13-PE versus CD36-APC on whole population; (right) CD34-perCP-Cy5.5 versus GFP (gated on CD13⁻ population). Numbers within plots represent percentage of cells within each quadrant; quadrants delimit background fluorescence of control-stained/mock-transduced cells. (B) Corresponding plots following enrichment of GFP⁺CD13⁻ cells (each plot shows whole population). Similar data were generated from control cultures expressing GFP alone (not shown). (C) Representative density plots showing expression of N-Ras protein and GFP in control (GFP alone) and N-Ras*–transduced day 7 erythroid cultures. Similar results were obtained from day-14 EPO-replete cultures (not shown). Note that fixation/permeabilization conditions were optimized for detection of the N-Ras antigen; the fluorescence of GFP was reduced by approximately 10-fold compared with fresh cells.