Figure 3. Binding and internalization of D7(VL-VH)-PE40 into PSMA positive cells.

C4-2 cells were incubated with immunotoxin at A. 4°C or B. 37°C, 5% CO₂, for 4 h. Then the immunotoxin was stained with mouse anti-human c-myc mAb and goat anti-mouse-IgG-AF488 (green). Early endosomes were labeled with rabbit anti-human early endosome antigen 1 (EEA-1) and anti-rabbit-IgG-DyLight®650 (red). Nuclei were stained with DAPI (blue). Images were taken by Confocal Laser Scanning Microscopy. D7(VL-VH)-PE40 bound at the cell surface at both temperatures. At 37°C the immunotoxin also localized within endosomes (yellow merge, yellow arrows) as well as in other compartments inside the cells (white arrow).