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. 2018 Jan 15;8:2689. doi: 10.3389/fmicb.2017.02689

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Copyright © 2018 Lin, Cai, Chen, Ye, Wu, Wang, Lv, Shang and Qu.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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EMSA analysis of PhoP with the putative promoter regions. His-tagged PhoP was purified and phosphorylated (PhoP-P) by incubation with 50 mM acetylphosphate. The putative promoter regions of the genes/operons phoPQ, mgtA, slyB, shf–rfbU-virK-msbB2, icsA, rstAB, yoaE, hdeAB, yrbL, yhiWX, pagP, and ipaH7.8 were PCR-amplified. DNA probes were purified and labeled with digoxigenin. Gel shift reactions were performed by incubating the labeled probe with increasing concentrations of PhoP-P (range, 0.16–1.6 μM). Lane 1 of each blot contained a no-protein control. All samples were electrophoresed on a non-denaturing polyacrylamide gel and blotted onto a nylon membrane. After incubation with anti-digoxigenin antibody, CSPD chemiluminescent reagent was added. The DNA fragment within the virA coding region was used as a negative control.