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. 2023 Jan 20;14:341. doi: 10.1038/s41467-023-35922-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Left panel; biotinylated DNA template was bound to streptavidin magnetic beads and incubated for 20 min with purified RNAPII and GTFs. After washes, NTPs were added to initiate RNAPII elongation for 45 min. Middle panel; Three conditions were used: in the absence of NTPs where PIC is formed, in the presence of NTPs in which RNAPII is in elongation, and after the chase of NTPs in which RNAPII is blocked from elongating. Right panel; the binding of different factors was evaluated by immunoblotting. The signals for rEXD2 were quantified and plotted in arbitrary units (au). The values are the means of three independent experiments (+/−SD) (Technical triplicates). Molecular sizes are indicated (KDa). Source data are provided as a Source Data file. b The biotinylated DNA template was bound to streptavidin magnetic beads and incubated for 20 min with purified RNAPII and GTFs with or without EXD2 as indicated. After washes, ATP or NTPs were added for 45 min. The binding of different factors was evaluated by immunoblotting and the signals for EXD2 were quantified and plotted in arbitrary units (au). The values are the means of three independent experiments (+/−SD) (Technical triplicates). Molecular sizes are indicated (KDa). Source data are provided as a Source Data file. c Number of nuclear GFP/RNAPII PLA foci in U-2 OS cells expressing either GFP or EXD2-GFP with or without Flavopiridol treatment (n = at least 100 cells per conditions from three independent experiments). Red bars indicate mean integrated density. One-way ANOVA with post-hoc Tukey adjustment comparisons were used to determine the p-values. d Left panel; rEXD2^WT and purified RNAPII were resolved by SDS-PAGE and immunoblotted with anti-EXD2 and anti-RPB1 antibodies. Molecular sizes are indicated (KDa). Right panel; Coomassie staining of recombinant EXD2^WT and EXD2^D108A/E110A. Lane 3 is the protein markers. Source data are provided as a Source Data file. e RNA (309nts) was transcribed from a linear template containing the AdML promoter using a reconstituted RNAPII run-off transcription assay. Then, increasing amount of recombinant EXD2^WT or EXD2^D108A/E110A (10, 20 and 30 ng) was added to the reaction for an additional 10 min incubation period before the reaction was stopped. Molecular sizes are indicated (Nucleotides). Source data are provided as a Source Data file.