Figure 2. Analysis of HIV clade C Gag protein expression.

HeLa cells were transfected with 2 µg of plasmid (pCMV-gagopt, pCMVi-gag, pCMVi-gagopt, or pCA-gagopt) (A) or infected with the corresponding Ad vector at MOI 5 (Ad-CMV-gagopt, Ad-CMVi-gag, Ad-CMVi-gagopt, or Ad-CA-gagopt) (B). Untreated HeLa cells were used as a negative control. After two days of incubation, the cells were harvested, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, transferred, and assayed by western immunoblotting using a Gag-specific Ab. To calculate the Gag protein relative intensity, the cell lysates from pCMVi-gag or Ad-CMVi-gag constructs were 10- or 50-fold more loaded in these lanes than in any of the other lanes, respectively. The number indicates the mean relative protein intensity to the Gag protein. Data are representative of five independent experiments. (C) Analysis of gagopt mRNA expression in HeLa cells infected with Ad-CMV-gagopt, Ad-CMVi-gagopt or Ad-CA-gagopt by quantitative real-time PCR. HeLa cells were infected with the corresponding Ad vector at MOI 2 (Ad-CMV-gagopt, Ad-CMVi-gagopt or Ad-CA-gagopt). After two days of incubation, total RNA was isolated from infected cells and cDNA was generated. cDNA was used as a template for quantitative real-time PCR. Data represent the mean ± standard error of the mean (S.E.M.) and are representative of two independent experiments. *, p<0.05; **, p<0.01.