Figure 5.

RabGAP activity and ARF binding ability are required for EspG activity.

A. Expression and secretion of EspG-R/Q:HA (RabGAP deficient) and EspG-E392R:HA (ARF binding deficient) proteins from ΔespG compared with EspG:HA.

B. Representative immunofluorescence images of the cellular localization of both complementation constructs compared with EspG:HA. Rab11–GFP expressing cells were infected for 5 h and stained with anti-HA antibodies and Phalloidin was used to detect actin pedestals which form under attached bacteria.

C and D. Surface levels of (C) TfR or (D) EGFR of cells infected for 5 h with the indicated strains in the presence of cycloheximide (20 μg ml⁻¹) were measured by addition of fluorescent ligands and quantified by flow cytometry. The MFI of surface-bound Tf-647 or EGF-488 is expressed as a percentage of the MFI of uninfected cells and the mean ± SEM of three independent experiments plotted.E. TfR recycling was measured by comparing the cell-associated fluorescence of cells which have endocytosed fluorescent Tf to those that have undergone unlabelled Tf chase for 10, 30 and 60 min. Data are expressed as ‘% retained Tf’ (Tf retained following chase/total Tf endocytosed). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.