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. 2019 Nov 13;10:2551. doi: 10.3389/fmicb.2019.02551

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Copyright © 2019 Tseytin, Mitrovic, David, Langenfeld, Zarivach, Diepold and Sal-Man.

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) and the copyright owner(s) 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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TMD-exchanged versions of EscS associate with the T3SS complex. Membrane protein extracts of ΔescD carrying pEscD-V₅ alone or in combination with pEscS_wt-HA, pEscS-TMD1_ex-HA, or pEscS-TMD2_ex-HA, were incubated in BN sample buffer and then subjected to BN-PAGE (upper panel), SDS-PAGE (lower panel) and western blot analysis using anti-V₅ and anti-HA antibodies. (Upper panel) BN-PAGE analysis showed that while the EscD-V₅ protein migrated mainly as a large complex at the top of the gel, the EscS WT and the exchanged TMD-versions migrated both as large and small protein complexes. (Lower panel) To confirm similar EscS and EscD expression among the samples, membrane protein extracts were analyzed by SDS-PAGE and western blot analysis using anti-HA and anti-V₅ antibodies. Similar protein expression levels were observed.