Figure 4. The ArnT C-terminus resides in the periplasmic space.

(a) Whole cells from E. coli strain DH5α carrying plasmids encoding cysteine-substitution of the C-terminal of ArnT were treated with buffer alone or 1 mM PEG-mal with or without 2% SDS for 1 h at room temperature and quenched with 45 mM DTT. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane for blotting with anti-FLAG to detected ArnT. (b) Immunoblot of total membranes from E. coli CC118 cells expressing _FLAG-ArnT-_PhoA performed with anti-FLAG. L, BLUeye prestained protein ladder. (c) Quantification of PhoA activity in CC118 E. coli expressing _FLAG-ArnT_PhoA. Cells were grown in LB and induced with 0.2% arabinose. Alkaline phosphatase activity was determined by measuring the rate of PNPP hydrolysis. pAH01 encodes _FLAG-Wzx-K367-_PhoA and pAH1801 encodes _FLAG-Wzx-T242-_PhoA, which were used as positive and negative controls, respectively³⁰. The values represent the mean ± SE from three independent experiments.