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. 2012 Oct 29;7(10):e47756. doi: 10.1371/journal.pone.0047756

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© 2012 Fengler et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Shown are immunoblot analyses utilizing anti-FLAG antibodies to monitor FLAG-tagged ToxR production of pFLAGtoxRS constructs, performed under reducing (panel A) and non-reducing conditions (panel B). pFLAGtoxRS was expressed in E. coli cells grown in LB broth to mid-log phase (OD₆₀₀ of 0.5) and subsequently induced with IPTG for 1 h. From left to right, shown are pFLAGtoxRS(Δ264), pFLAGtoxRS and pFLAGtoxR^CCS, either containing no ToxR operator sequence or ompU or toxT operator sequences, respectively. A 55 kDa ToxR cross-reacting protein band, associated with pFLAGtoxRS and pFLAGtoxR^CCS, is indicated by an arrow. To note, cysteinyl dependent homodimer and oligomer ToxR bands occurred diminished as observed for pFLAGtoxRS in comparison to pFLAGtoxRS(Δ264). Molecular size markers are indicated on the left. Immunoblot analysis was performed at least three times, and results were reproducible.