Fig. 5. Cells in G2 (cyclin B1 positive) and anaphase bind preferentially to Shiga toxin. (a,b) Non-synchronized Vero cells were exposed to Cy2-ST and Cy3-CTX and analyzed by fluorescence microscopy as in Fig. 1a. Cells were then fixed, permeabilized and immunostained for cyclin B1 (see Materals and Methods section). (a) and (b) represent different experiments of the same kind. Overlay represents the overlay of CTX, ST and cyclin B1. (c) FACS-separated cells, binding either ST (lane 1) or CTX (lane 2) were extracted and identical amounts of proteins separated by SDS-PAGE. Immunoblots were performed using anti-cyclin-B1- and anti-protein disulfide isomerase antibodies. Lane 3 represents extracts from the same cells but before FACS separation. The amount of protein in lane 3 was about four times that applied in lanes 1 and 2. (d) A cell in anaphase shows a significant binding and uptake of ST but not of CTX. Chromatin staining with DAPI. The cells were fixed about 10 minutes after start of uptake of the toxins. At this time point significant amounts of the toxins have already entered the cells.