Figure 2

EPOR expression in CG4-EPOR cells. (A) CG4 cells were transduced with a bicistronic lentiviral vector expressing EPOR and EGFP and cultured in GM. The efficiency of transfection in genetically modified (CG4-EPOR) cells was verified by measuring by flow cytometry the EGFP reporter expression, whose translation is linked to EPOR via the internal ribosome entry site (IRES). About 90% of the CG4-EPOR cells expressed EGFP, as opposed to 1.4% of background fluorescence in wild-type CG4 cells. The experiment is representative of three experiments. EGFP expression in single experiments was 83%, 90.2% and 87.9%. (B) CG4-EPOR were switched to DM and cultured for 3, 6 and 9 d with or without EPO. EPOR expression during cell differentiation was confirmed by immunoblotting with anti-V5-tag mouse antibody. As a negative control, wild-type CG4 cells at d 9 of differentiation also were analyzed for transduced EPOR expression. The numbers on the left of the panel indicate the positions of molecular weight markers of the indicated sizes in kDa.