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Fig. 2. Rip11/FIP5 knockdown increases Tf uptake. (A) HeLa cells treated with mock siRNA, Rip11/FIP5 siRNA1 or Rip11/FIP5 siRNA2 were incubated at 37°C with Tf-Alexa488. Cells were then washed, fixed and internalized Tf-Alexa488 measured by flow cytometry. The data shown are the mean ± s.d. of three independent experiments. Asterisks indicate time points that are significantly different from mock-treated control at P<0.025. (B) Mock-treated or Rip11/FIP5 siRNA-treated HeLa cells were fixed and incubated with 80 µg/ml Tf-Alexa488 in the absence or presence of 0.4% saponin. Cells were then washed and plasma-membrane-associated (non-permeabilized cells, see figure) and total cellular (permeabilized cells, not shown) Tf-Alexa488 measured by flow cytometry. The data shown are the mean ± s.d. of three independent experiments. (C) Mock- and Rip11/FIP5 siRNA1-treated HeLa cells were incubated at 37°C for 30 minutes with Tf-Alexa488. Cells were then washed and incubated at 37°C with unlabeled Tf. Cell-associated Tf-Alexa488 was measured by flow cytometry and is shown as arbitrary units. The data shown are the mean ± s.d. of three independent experiments. *P<0.01. (D) The rates of Tf uptake and recycling calculated from the data presented in A and C. The data shown are the half-life of uptake and recycling (T1/2) as well as the amount of Tf-Alexa488 internalized and recycled (arbitrary fluorescence units per minute). Data are the mean ± s.d. calculated from three different experiments. *P<0.02.
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