Effect of 2APro expression on protein synthesis via Cap or IRES structures.

<p>A. HeLa cells were co-transfected with the Luciferase expression vector pGL3 and pU2A in different ratios (1∶3; 1∶1; and 3∶1). The total amount of DNA in each transfection was adjusted with the addition of the pcDNA3.1 empty vector. Cells were lysed after 48 or 72 hours, Luciferase substrate was added and light was measured using a luminometer. B. HeLa cells were co-transfected with the cap-dependent eGFP expression vector and pU2A in different ratios (1∶3; 1∶1; and 3∶1). The total amount of DNA in each transfection was adjusted with the addition of the pcDNA3.1 empty vector. Cells were fixed after 72 hours of transfection and the expression of eGFP was analyzed by the emission of green fluorescence. Left column represents the bright field. C. HeLa cells were co-transfected with the HIV-1 infectious clone pBR43IeG, which express the eGFP reporter gene under the control of an IRES element and pU2A in different ratios (1∶3; 1∶1; and 3∶1). The total amount of DNA in each transfection was adjusted with the addition of the pcDNA3.1 empty vector. Cells were fixed after 48 or 72 hours and GFP expression was analyzed by flow cytometry. The percentage of eGFP positive cells was plotted. D. Geometric mean of GFP emission analyzed by flow cytometry at C. p values <0.05 were considered significant and are marked with an asterisk.