FIGURE 10.

Effect of EGF on association of ARAP1 and mutants with reduced PtdIns(3,4,5)P₃ binding with a Rab5-positive endocytic compartment. A, images of representative cells expressing FLAG-ARAP1. HeLa cells expressing N-terminal FLAG-tagged ARAP1 or ARAP1 mutants and GFP-Rab5 were serum-starved overnight on fibronectin-coated coverslips (10 μg/ml) and treated with 100 ng/ml EGF for 5 min at 37 °C. Fixed cells were immunostained with monoclonal anti-FLAG antibody (red). Scale bar, 10 μm. B, quantification of colocalization of ARAP1 and mutants with Rab5-positive endosome. Effect of mutations, which affect binding to PtdIns(3,4,5)P₃, in colocalization of ARAP1 with Rab5. Pearson's coefficients for colocalization of ARAP1 and Rab5 were determined as described under ”Experimental Procedures“ for at least 30 cells in each condition.