Figure 2

Clathrin independent entry of the ricinB:QDs into HeLa cells. HeLa cells were subjected to vector-based siRNA knock-down of clathrin heavy chain for 3 days. The ricinB:QD655 bioconjugates were allowed to be internalized into the cells at 37°C for 30 min and 180 min, respectively (representative images, upper/lower panel). Alexa555 (al555)-transferrin (Tf) (green) was added to cells for the last 20 min of the endocytosis period. The cells were then fixed and prepared for confocal immuno-fluorescence microscopy labeling them with antibodies against clathrin heavy-chain, EEA1 and CD63, and with the appropriate secondary antibody-Cy2 (blue)/Cy5 (white) fluorophore-conjugates. Images show normal uptake of ricinB:QD655 in cells were clathrin is knocked down (− clathrin, no blue staining), whereas the alexa555-Tf uptake was inhibited and only weakly stained the periphery of these cells.