Figure 4.

Effects of Rififylin-GFP on Tf recycling. (A–D): HeLa cells were transfected with Rffl-GFP–expressing vector. Sixteen hours later, the cells were washed and incubated for 30 min in medium without serum at 37°C. The cells were then allowed to internalize Alexa⁵⁹⁴-Tf for 90 min at 37°C. The cells were either fixed at this point (0 min; A) or incubated for 30 min (B), 60 min (C), and 240 min (D) at 37°C. The cells were then fixed, stained with DAPI, and observed under a fluorescence microscope. Bars, 10 μm. (E) HeLa cells were transfected with expression vectors coding for Rffl-GFP or GFP as control. Sixteen hours later, the cells were washed, incubated for 30 min in medium without serum at 37°C, and incubated with Alexa⁶³³-Tf for 60 min at 37°C. The remaining surface-bound Alexa⁶³³-Tf was then removed by acid wash at 4°C. The cells were either kept on ice (0 min) or incubated for various times at 37°C. The intracellular associated fluorescence due to Alexa⁶³³-Tf retained after each chase time was then quantified by flow cytometry, gating for transfected (GFP-positive) cells. The graph shows the average ± SD of three independent experiments.