Fig. 10. GRIF1 overexpression inhibits trafficking of EGF-EGFR complexes from early endosomes to the lysosomal pathway. (A) HeLa cells transfected with pEGFP-GRIF1 were allowed to internalize TR-EGF for 10 minutes and then chased for 3 hours at 37°C. Cells were stained with a primary antibody against EEA1 and a CY5-conjugated secondary antibody. Arrowheads and asterisks indicate transfected and untransfected cells, respectively. Small arrows indicate EEA1-positive early endosomes that contain EGF. Merged images (bottom) compare the labeling of GRIF1 (green) with EGF (red), GRIF1 (green) with EEA1 (blue), and EGF (red) with EEA1 (blue). Bars, 10 µm. (B) HeLa cells transfected with the indicated GRIF1 constructs or the empty vector were allowed to internalize ¹²⁵I-EGF for 10 minutes and then chased for 1 hour at 37°C. The degraded ¹²⁵I-EGF is expressed as a percentage of the initially internalized ¹²⁵I-EGF. Values are mean ± s.e.m. from three independent experiments. The asterisks indicate a statistically significant difference (P<0.05) from the vector-transfected cells.