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. 2004 Mar;24(6):2444–2454. doi: 10.1128/MCB.24.6.2444-2454.2004

FIG. 2.

FIG. 2.

Inhibition of transcription elongation directed by the A20 promoter is mediated by DSIF. (A) Left panel: immunoblot analysis of Jurkat cell nuclear extract immunodepleted of DSIF p160 by using anti-DSIF p160 antibody. Anti-Oct-2 antibodies were used as the specificity control for the immunodepletion. Ten percent of precipitated proteins was loaded in lanes 4 and 5. Right panel: analysis of Pol II in DSIF-depleted extract. Shown are the results of immunoblot analysis of nuclear extract that was either untreated (lane 1) or immunodepleted of DSIF (lane 2) by using anti-DSIF p160 antibody and anti-Pol II antibody (large subunit). The beads (lane 3) represent the immunoprecipitated complex. (B) Transcription activity of the DSIF p160-depleted extract. Shown are the results of in vitro transcription reactions with either A20-lacZ or MLP U-less templates and Jurkat cell nuclear extract that was untreated (lanes 1 and 2) or immunodepleted with the indicated antibodies. The A20 transcripts were analyzed by primer extension assay with primers corresponding to +30, +60, and +120 relative to the transcription start site. The MLP transcript was analyzed by the run-on assay. In lanes 2, 4, 6, and 8 Sarkosyl (0.2%) was added 1.5 min after the addition of nucleotides. In lanes 7 and 8 the beads containing immunoprecipitated and washed DSIF were added back to the DSIF-depleted extract.