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. 2015 Sep 17;35(20):3459–3470. doi: 10.1128/MCB.00601-15

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FIG 3 — Human Rtf1 promotes transcription elongation independently of the PAF1C and DSIF. (A) Immunoblot analysis of HeLa cell NE after immunodepletion of one of the indicated factors. Mock-depleted NE (NEΔIgG) were analyzed as a control. The asterisk denotes a nonspecific signal. (B) Immunoblot analysis of the P0.3 D0.3 fraction after immunodepletion of Leo1 and/or DSIF. Mock-depleted P0.3 D0.3 (P0.3 D0.3 ΔIgG) was analyzed as a control. (C) In vitro transcription assays for immunodepleted P0.3 D0.3 fractions. Promoter-proximal and -distal products are indicated by the arrows. The amounts of promoter-proximal and -distal products and their ratios are shown below. (D) Immunoblot analysis of the P0.3 D0.3 fractions immunodepleted with anti-Leo1, anti-Cdc73, and anti-Paf1 antibodies (P0.3 D0.3 ΔPAF1C) or with control IgG. (E) The indicated amounts of P0.3 D0.3 fractions were added with or without Flag-Rtf1 to in vitro transcription assay mixtures. (F) A partially purified Rtf1 coactivator fraction derived from P0.3 D0.3 (P0.3 D0.3 H0.4 S number 5) was added, together with increasing amounts of Flag-Rtf1, to in vitro transcription assay mixtures. The amounts of promoter-proximal and -distal products and their ratios are shown below. (G) Immunoblot analysis of TFIIS and histones H2B and H3.