FIG 4 .
EscR TMD3 is critical for the T3SS activity. (A) Protein secretion profiles of WT, ΔescN, ΔescR, and ΔescR complemented with EscRWT-3HA or EscR-TM3ex-3HA EPEC strains grown under T3SS-inducing conditions. The secreted fractions were treated and analyzed as described in the Fig. 1A legend. Replacement of the TMD3 sequence with a 7L9A sequence completely abolished T3SS activity. The expression of EscRWT-3HA and EscR-TM3ex-3HA was examined by analyzing the bacterial pellets by SDS-PAGE and Western blot analysis with an anti-HA antibody. (B) The ΔescR EPEC strain carrying either EscRWT-3HA or EscR-TM3ex-3HA vector was grown under T3S-inducing conditions and was fractionated into periplasmic (P), cytoplasmic (C), and membrane (M) fractions. The samples were separated on an SDS-PAGE gel and analyzed by Western blotting using anti-HA antibody. To confirm correct bacterial fractionation, the Western blots were probed with anti-MBP (periplasmic marker), anti-DnaK (cytoplasmic marker), and anti-intimin (membrane marker) antibodies.