Fig. 8. Effect of EEA1 knockdown on Tf and EGF trafficking. (A) Real time quantitative PCR in HeLa cells stably expressing a scrambled control (C) or an EEA1-directed (KD) shRNA. The mRNAs examined are indicated along the x-axis. (B) Western blotting of EEA1 and TfR in two independent stable clones, and immunofluorescence analysis of EEA1. (C) Cells were serum starved and incubated in the presence of EGF for the times indicated. Extracts were analyzed by western blotting with anti-EGFR antibodies. Values are mean ± s.e.m. of four independent experiments analyzed by densitometric scanning. (D) Kinetics of Tf uptake and recycling in C and KD cells. Values are mean ± s.e.m. of three experiments performed in duplicate.