10.1371/journal.pone.0105749.g008 Tomotaka Tanabe Tomotaka Tanabe Ayaka Kato Ayaka Kato Keiichi Shiuchi Keiichi Shiuchi Katsushiro Miyamoto Katsushiro Miyamoto Hiroshi Tsujibo Hiroshi Tsujibo Jun Maki Jun Maki Shigeo Yamamoto Shigeo Yamamoto Tatsuya Funahashi Tatsuya Funahashi <i>In vitro</i> translation of <i>peuA</i> mRNA. Public Library of Science 2014 pH 8.0 pH 7.0 translation stage PeuA complementation experiments Secondary structure prediction Vibrio parahaemolyticus peuA Gene Encoding silico analyses Primer extension analysis Vibrio parahaemolyticus ferric uptake regulator TonB 2 system genes translation analysis deletion mutants peuRS deletion novel gene regulation mechanism enterobactin utilization system operon undescribed receptor peuA expression site vpa transcript Translation initiation Alternative Ferric Enterobactin Receptor system PeuRS 2014-08-22 03:34:55 Figure https://plos.figshare.com/articles/figure/_In_vitro_translation_of_peuA_mRNA_/1149419 <p>(A) <i>In vitro</i> translation analysis of the +1 and +39 <i>peuA</i> transcripts labeled with the FLAG tag. The +1-<i>peuA</i>′-<i>flag</i> RNA and +39-<i>peuA</i>′-<i>flag</i> RNA were first synthesized by <i>in vitro</i> transcription, as described in the MATERIALS AND METHODS, and a mixture containing either the +1-<i>peuA</i>′-<i>flag</i> RNA (30 pmol)/<i>fur</i>-<i>flag</i> RNA (3 pmol) or the +39-<i>peuA</i>′-<i>flag</i> RNA (30 pmol)/<i>fur</i>-<i>flag</i> RNA (3 pmol) as the template was subjected to <i>in vitro</i> translation. The FLAG-fused proteins translated were separated on 15% SDS-polyacrylamide gels, and were detected by western blotting using anti-FLAG IgG. (B) Confirmation of the presence of +1-<i>peuA</i>′-<i>flag</i> RNA and +39-<i>peuA</i>′-<i>flag</i> RNA in the reaction mixture for <i>in vitro</i> translation. These RNA fragments were detected in the reaction mixture by northern blotting using a DIG-labeled <i>peuA</i> probe.</p>