Figure 3
eYfnI export and release are independent of its phosphorylation state. (A) Localization of YfnI-FLAG by western blot experiments using anti-FLAG antibodies. Strains expressing yfnI-flag in a WT, ΔprkC or ΔprpC genetic background were grown in LB medium containing 0.5% xylose until the end of exponential phase. Subsequently, protein extracts from culture supernatants or bacterial pellets were prepared and separated by SDS-PAGE and the FLAG-tagged proteins visualized by western blot using anti-FLAG antibodies. The eYfnI-FLAG domain was only detected in the culture supernatants for all three strains tested. (B) Determination of the localization of WT, phosphomimetic (T297D) and phosphoablative (T297A) YfnI-FLAG by western blot analysis. Protein extracts from culture supernatants or bacterial pellets from strains grown in LB medium containing 0.5% xylose until the end of exponential phase and expressing yfnI-T297D-flag, yfnI-T297A-flag or yfnI-flag were prepared and separated by SDS-PAGE and the FLAG-tagged proteins detected by western blot using anti-FLAG antibodies. The extracellular domains of the three proteins were only detected in the culture supernatant fractions. Coomassie stained gels showing the amount of proteins in the extracts used in these western blots are presented in Fig. S3. Specificity of anti-FLAG antibodies is shown in Fig. S4.
