Figure 8.

Kinetic analysis of the effect of microinjected anti-Glu tubulin antibody on Tf recycling. TRVb-1 cells were incubated with Cy3-Tf for 1 h. Cells were then microinjected with anti-Glu tubulin antibody followed by a chase in the absence of Cy3-Tf but with excess unlabeled Tf and desferroxamine for the indicated times before fixation. Alexa488-BSA was included in the injection solution to mark microinjected cells. As described in the MATERIALS AND METHODS section, after microinjection, cells were rinsed three times in medium 1 (5 min) before Tf chase. Images of cells (both injected and uninjected) were collected at the indicated time points, and the fluorescence power of Cy3-Tf per cell was measured. Results of a representative example of all the experiments are shown. Error bars represent the SD and 100% represents the fluorescence power of Cy3-Tf at 0 min chase after the initial pause labeling of Cy3-Tf.