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. 2000 Jun 13;97(13):7184–7189. doi: 10.1073/pnas.140202297

Figure 1.

Figure 1

Immunodepletion of NC2 selectively reduces activated transcription in HeLa NE. Transcription reactions (20 μl) contained 25 μg NE proteins, 50 fmol of each promoter template, and 20 ng of recombinant GAL4-VP16. Transcripts were analyzed by primer extension and visualized by autoradiography and PhosphorImaging. The positions of primer extension products corresponding to correctly initiated transcripts are indicated. Relative amounts of specific transcripts and fold activations are based on PhosphorImager quantitation. (A) Schematic representation of promoter constructs used in this study. (B) Multiple-round transcription reactions were carried out for 60 min in the presence of NTPs (*). Strong enhancer-dependent HIV-1 transcription occasionally resulted in the generation of additional bands that obscured transcripts from the enhancer-less HIV-1(−33/+68) promoter template (B, lane 1). In these cases, fold activation of transcription was calculated from absolute amounts of HIV-1(−33/+68) basal transcription observed in the presence of the G5HIV(−33/+80) construct in the absence of GAL4-VP16 (B, lanes 3 and 4). (C) Single-round transcription reactions were stopped 1 min after addition of NTPs.