Figure 8.

Effect of RCP knock-down on TfR endocytosis, lysosomal degradation and recycling. (A) HeLa cells were either mock transfected or transfected with Rip11 or RCP siRNAs, and Triton X-100 extracts of them were analyzed by Western blotting with anti-RCP, anti-Rip11, anti-Rab11, and anti-γ adaptin antibodies to determine the extent and specificity of the knock-down. (B) HeLa cells were either mock transfected (control) or transfected with Rip11 or RCP siRNAs. After 72 h, cells were incubated for 30 min at 4°C with 20 μg/ml Tf-Alexa647. Cells were then incubated for additional 30 min at 37°C in the presence of Tf-Alexa647. Cells were then washed, fixed in 3% paraformaldehyde, and amounts of internalized Tf-Alexa647 were quantitated by FACS. The data presented are means ± SE of three independent experiments. (C) HeLa cells were either mock transfected or transfected with Rip11 or RCP siRNAs. After 72 h, cells were incubated for 30 min at 4°C with 2 μg/ml anti-TfR-PE antibody. The amount of plasma membrane bound anti-TfR-PE antibody is shown in the inset. Cells were then moved to at 37°C and incubated for varying amounts of time in the presence of 20 μg/ml Tf-Alexa647. Cells were then washed, fixed in 3% paraformaldehyde, and the time course of anti-TfR-PE antibody internalization was quantitated by FACS analysis. (D) HeLa cells were either mock transfected (control) or transfected with Rip11 or RCP siRNAs. After 72 h, cells were incubated for 30 min with Tf-Alexa647 at 4°C followed by additional 20-min incubation at 37°C. Cells were then washed and incubated at 37°C for 20 min in the presence of unlabeled Tf. The amounts of cell associated Tf-Alexa647 was quantitated by FACS. The data presented are means ± SE of three independent experiments.