Fig. 6: Cy3B-labelled LifA clusters at the membrane and is internalised by bovine T-lymphocytes.
From: Structure of lymphostatin, a large multi-functional virulence factor of pathogenic Escherichia coli
LifA formed clusters (white arrows) at the plasma membranes of bovine T-lymphocytes following incubation with Cy3B-labelled LifA. With increasing time following addition of LifA-Cy3B a greater percentage of clusters were found within T-lymphocytes distal to the cell membrane. Panels a–d show representative confocal z-section images at 20, 30, 45 and 60 min after addition of LifA-Cy3B, respectively. Cells were incubated with LifA-Cy3B (magenta) before fixing at the intervals indicated and staining with WGA633 (blue) and DAPI (cyan). For each time point at least two images were taken at a similar magnification (n ≥ 2) as shown in panel a–d. At least eight images were taken per time point at a smaller magnification (n ≥ 8) to show several cells per image for counting the clusters. Panel e shows the percentage of LifA-Cy3B clusters proximal or distal (by at least 500 nm) to the plasma membrane over time. The location of at least 100 clusters in z-stack images was analysed at each time point. Source data are provided as a Source Data file.
