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. 2005 Mar 1;33(4):1269–1279. doi: 10.1093/nar/gki274

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© The Author 2005. Published by Oxford University Press. All rights reserved

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Immunodepletion of NELF impairs promoter proximal pausing in vitro. (A) Permanganate footprinting analysis of mock-depleted and NELF-depleted extracts. Transcription reactions were done in the absence of nucleotides (lanes 1, 4, 7 and 10), the presence of ATP, CTP, UTP and 3′ O-methyl GTP (lanes 2, 5, 8 and 11), or the presence of ATP, CTP, UTP and GTP (lanes 3, 6, 9, and 12). At the end of 30 min, Pol II was detected on the DNA by permanganate footprinting. Permanganate reactivity at +8 and +22 in lanes 2, 5, 8 and 11 was due to Pol II arresting at +15 following incorporation of the 3′ O-methyl GTP. Permanganate reactivity at +22, +30 and +34 in lanes 3 and 9 was due to Pol II stably paused in the promoter proximal region of hsp70. (B) Quantification of band intensities detected at +22 and +30 in lanes 3, 6, 9 and 12. Background intensities detected in lanes 1, 4, 7 and 10 have been subtracted. (C) Immunoblot analysis showing the specificity of the NELF depletions. Depleted extracts were run-on a 4–15% gradient gel and blotted to nitrocellulose. The largest subunit of DSIF and the indicated subunits of NELF were detected with antibodies.