Effect of hSpt5 siRNA on HIV-1 Tat transactivation in Magi cells. Quantified effect of siRNA on HIV-1 Tat transactivation was determined by measuring β-galactosidase activity. Magi cells were co-transfected with pTat-RFP plasmid and various siRNAs targeting hSpt5 or Tat and harvested at 48 h post-transfection. Activity of β-galactosidase was measured using the β-Galactosidase Enzyme Assay System (Promega). Tat transactivation was determined by the ratio of β-galactosidase activity in pTat-RFP transfected cells to activity measured in cells without pTat-RFP. The inhibitory effect of siRNA was determined by normalizing Tat transactivation activity to the amount of Tat-RFP protein. Tat transactivation was measured for Magi cells transfected with pTat-RFP only (lane 1), or Tat-RFP transfected with single-stranded antisense hSpt5 siRNA (hSpt5-AS, lane 2), hSpt5 duplex siRNA (hSpt5-DS, lane 3), mismatch hSpt5 duplex siRNA (hSpt5-mm-DS, lane 4), or Tat siRNA duplex (Tat-DS, lane 5). Results are representative of three independent experiments.