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. 2012 Mar 30;7(3):e34039. doi: 10.1371/journal.pone.0034039

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Complementation plasmids were introduced into each mutant and the KIM5 parent, and the strains were evaluated by performing secretion assays. The TTSS mutant, yscU, was included as a negative control. Y. pestis strains were grown in MM9 at 26°C for 2 hours and then shifted to 37°C for 3 hours to induce secretion. Proteins in the supernatant (S) and pellet (P) fractions were precipitated and visualized by immunoblotting with antibodies to YopM, YopE (TTSS secreted proteins), YscD (TTSS apparatus component) and RpoA (RNA Polymerase alpha subunit, loading and fractionation control). An asterisk indicates truncated Yops due to degradation by Pla protease.