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. 2020 Dec 3;48(22):12858–12873. doi: 10.1093/nar/gkaa1163

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Immunologic and enzymatic detection of PolI in active MTase fractions from in vivo expression. Panel (A): Anti-His-tag detects 6xHis::M.BceJIII from pAF9 in both active and further-purified inactive fractions (top row), but anti-PolI detects the polymerase only in the MTase active fraction (bottom row). Panel (B): MTase action and nucleotide incorporation by these fractions. Blue bars: single-stranded M13mp18 DNA modified with [H³]SAM; red bars: sperm whale DNA labeled by [H³] dTTP (DNA polymerase activity) measured without enzyme (1) or with active (2) or inactive (3) MTase column fractions. Panel (C): Reciprocal immunoprecipitation assays recover PolI and His-tagged MTases together. Top panel: Western detection of PolI by anti-PolI of MTase tagged anti-His IP. Bottom panel: Western detection of His-tagged MTase anti-His of anti-PolI IP. In vivo expression employed pAF9 (M.BceJIII), pAF10 (M.EcoGIX WT) and pAF11 (M.EcoGIX mut).