Fig. 6. Suppression of PIKfyve does not affect endosomal sorting of the EGF receptor. HeLa cells were treated with either control, PIKfyve specific siRNA duplex II or a combination of PIKfyve specific siRNA duplexes II and V for 72 hours. (A) Cells were serum-starved for 3 hours, labelled with ¹²⁵I-EGF at 1 kBq per well for 1 hour at 4°C, allowed to internalise surface-bound ¹²⁵I-EGF for 5 minutes at 37°C and were then returned to 4°C. Cells were chased using medium containing 100 ng/ml unlabelled EGF for various times at 37°C. Recycled, degraded and internalised fractions were subjected to -radiation counting. (B) Serum-starved cells were stimulated with 100 ng/ml EGF for 0, 10, 20, 30, 60 and 120 minutes, and western blotted with antibodies against EGF receptor or tubulin (loading control). (C) Quantification of data from three independent experiments.