Tandem PxxP motifs are required for EspFU recruitment and pedestal formation by EHEC. FLCs were infected for 3.5 h with strain EPEC KC12 expressing the EspFU HP (R475), PHP, PHPP28/31A, and PHPP36/39A variants and were visualized using fluorescent microscopy after staining with DAPI to localize bacteria (blue), FITC–anti-myc antibody was used to detect EspFU derivatives (green), and Alexa568-phalloidin was used to detect F-actin (red).