Fig. 9. Overexpression of GRIF1 does not affect the endocytosis of EGF or transferrin. (A,B) HeLa cells transfected with pEGFP-GRIF1 (green) were treated with TR-transferrin for 30 minutes (red; A) or with TR-EGF for 10 minutes (red; B) at 37°C. Cells were then examined by immunofluorescence microscopy. Arrowheads and asterisks indicate transfected and untransfected cells, respectively. Bars, 10 µm. (C) Schematic representation of the GRIF1 deletion mutants used in the ¹²⁵I-EGF endocytosis assays. The Hrs-binding domain (residues 359-507) and the kinesin-binding domain (residues 128-283) are indicated by brackets. (D) HeLa cells were transiently transfected with empty vector (CTL) or the following GRIF1 constructs: WT (GRIF1), 1 (GRIF11), 2 (GRIF12), 3 (GRIF13), 4 (GRIF14). Cells were incubated with ¹²⁵I-EGF for 10 minutes at 37°C. The internalized ¹²⁵I-EGF is expressed as a percentage of the initially bound ¹²⁵I-EGF. Values are mean ± s.e.m. from three independent experiments.