Tax opposes HBZ-mediated activation of Dkk1 expression. (A) Dkk1 expression in uninfected and HTLV-1-infected T-cell lines. Levels of Dkk1 and UBE2D2 mRNA from indicated cell lines were analyzed by RT-PCR. + RT and -RT denote cDNA synthesis with and without reverse transcriptase, respectively. (B) Levels of Dkk1 mRNA in cells transfected with the indicated expression vectors. Values were determined as described in the legend of Figure 1B, using data from at least three independent transfections. Lower panels show a Western blot analysis of Tax and β actin from cellular lysates (40 μg) prepared from one set of transfected cells. (C) Levels of Dkk1 in culture media from cells transfected with pSG5 or Tax. Acid-precipitated proteins from culture media (upper panel) and 30 μg of cellular lysates (lower panels) were subjected to western blot analysis. (D) Levels of HBZ and Tax expression in HTLV-1-infected T-cell lines. HBZ and Tax mRNA copy numbers were normalized to the number of UBE2D2 mRNA copies following quantitative real-time PCR and construction of a standard curve for mRNA copy number determined by amplification of 10-fold serial dilutions of the pSG-THU plasmid that contains the Tax, HBZ and UBE2D2 amplification targets. The graph shows data from at least two independent RNA extractions from each cell line. The average mRNA copy numbers for Tax and HBZ relative to UBE2D2 are indicated below each cell line.