Validation of differentially expressed genes at the RNA and protein levels in bovine 23CLN cells. 23CLN cells were transfected with pCAGGS encoding Flag-tagged Tax proteins (TaxWT, open column; TaxS240, shaded column; TaxD247G, black column) or the control pCAGGS (light column). Thereafter, 24 h after transfection, cells were subjected to confocal laser-scanning analysis to detect localization (A) and to real-time RT-PCR ( B and C) and western blotting ( D) to validate in bovine cells differential expression of cellular genes had altered after expression of wild-type and mutant Tax proteins in HeLa cells. The bars indicate the fold change expression of up- ( B) or downregulated ( C) genes regulated by Tax. Data were normalized to GAPDH mRNA expression. The results represent the mean of 2 samples from 1 experiment. ( D) The protein expression of Tax and Fos, Rrad, and Tnfaip6 was validated by western blotting. Actin was used as a control.