Comparison of transgene expression levels in A549 cells infected with monovalent Ad5 vectors. A549 cells were infected 48 hours post plant with various concentrations of monovalent Ad5 vectors in α-MEM media supplemented with 10% heat-inactivated FBS and 5% Pen/Strep. The cells were placed at 37°C/5% CO2 for 48 hours and then lysed with either a sodium deoxycholate/IGEPAL buffer for ELISA analysis or an RNA extraction buffer (RNeasy 96 Qiagen kit). The cell lysates were denatured and reduced prior SDS-PAGE analysis. Gag, pol and nef proteins were detected by colorimetric ELISA using HRP/TMB detection (A), gag, pol and nef mRNAs were detected by RT-qPCR (B). The protein profile of pol and nef proteins obtained by silver-stained SDS-PAGE is shown in (C). The MOI was determined by the quantity of infectious Ad5 particles measured by quantitative PCR based potency assay along with total Ad5 particles as measured by a genome quantitation assay. The error bars represent 95% confidence interval.