Figure 5. C-terminus of ArnT from S.

enterica is located in the periplasm. (a) Immunoblot of total membranes isolated from E. coli DH5α containing plasmids expressing ArnT_Se cysteine replacements. The proteins were separated by SDS-PAGE and immunoblotting was performed using anti-FLAG. ArnT_{Se-FLAG-10xHis} was used as control. C148A-C149A (CC/AA) was used as a template to generate the cysteine-substitution in the C-terminal of ArnT_Se. (b) PEGylated samples of ArnT_Se were prepared and analyzed as described in the legend to Fig. 2B. The positions of the molecular mass marker are indicated to the left. Asterisks indicate PEGylated ArnT_Se forms. ArnT_Se aggregates at the top of the western blots are attributed to the mild denaturing conditions. (c) Immunoblot of total membranes from E. coli CC118 cells expressing _FLAG-ArnT_Se-PhoA performed with anti-FLAG. (d) Quantification of PhoA activity in CC118 E. coli expressing _FLAG-ArnT_Se-PhoA. Cells were processed as described in the legend to Fig. 4C using the same positive and negative controls. The values represent the mean ± SE from three independent experiments.