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Fig. 3. Transgene expression in transfected MCF-7 cells. MCF-7 cells were transfected with bicistronic expression vectors [RAR and EGFP under the control of the cytomegalovirus (CMV) promoter], stable transfectants selected in the absence of RA, and EGFP protein assayed to infer RAR expression levels. A, Western blot analysis showing similar overall EGFP expression levels after stable transfection with equal amounts (2 µg) of pRAR{alpha}2-IRES-2-EGFP and pRARß2-IRES-2-EGFP. To verify that the CMV promoter was not RA dependent, EGFP expression was also monitored after 24-h treatment with 1 µM RA; this had no effect on EGFP expression, as expected. B, fluorescence microscopy (EGFP autofluorescence) of MCF-7 cultures transfected as in A and, as a control, transfected with 2 µg of pIRES-2-EGFP vector. The fluorescent signals produced by the three cell groups are similar.