Differentiation of human keratinocytes influences SFK protein and mRNA expression in transduced HFK cells. (A) Western blot analysis of differentiating human foreskin keratinocytes (HFK) transduced with HPV 16 E6, E7 or both oncogenes. Cellular differentiation was induced by culturing cells in the presence of serum and high calcium. Levels of cellular p53 and Rb proteins are reduced in the presence of functionally active HPV 16 E6 and E7 oncoproteins, respectively. (B) Analysis of Src, Yes, and Fyn protein expression by western blot using antibodies specific for the native form of the kinases and for a C-terminal tyrosine residue that is phosphorylated upon activation. Actin was used as loading control. The results shown are representative of three independent experiments. (C) The effect of HPV 16 oncogenes on the relative mRNA expression of Src, Yes, and Fyn in differentiating HFK cells. Real-time PCR analysis was performed using RNA purified from the transduced HFK cells. The values were normalized against GAPDH mRNA and presented as relative levels with respect to the value of cells transduced by LXSN. The bars represent the mean ± SEM of two independent experiments, each with triplicate measurements. Two-tailed Student t-test was carried out for the analysis of mRNA expression levels to determine statistical significance (P < 0.05).