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Fig. 5. Effects of BPGAP1 and EEN on EGF receptor endocytosis. HeLa cells were untransfected (A) or transfected with different epitope-tagged expression plasmids coding for wildtype BPGAP1, PP, R232A and/or wildtype EEN and NT, either alone (B) or in combination (C) as indicated. Cells were then made quiescent by serum-removal for 2 hours, followed by treatment with 100 ng/ml EGF for 10 minutes in the presence of monensin. Cells were permeabilized, stained and visualized under confocal fluorescent microcopy as described in Materials and Methods. Untransfected cells in A were labeled with rhodamine-phalloidin to mark the shape of cells and the intracellular vesicles (A-C) indicate uptake of EGF receptor (grey). Cells expressing different constructs of BPGAP1 (red) and EEN (blue) were detected by appropriate anti-Flag, anti-HA or anti-Myc antibodies followed by fluorophore-conjugated secondary antibodies. Images from cells transfected with multiple constructs were merged (Merged¹) to show their colocalization (purple). The second merged images (Merged²) illustrate the endocytosed EGF receptor in transfected cells. For the triple transfectants (asterisk), the transfection was optimized to express the wildtype BPGAP1 in all the cells that expressed PP mutant, validated by parallel experiments (data not shown). The intensities of images were enhanced to capture changes in the cell peripheries, including their cell protrusions. Bars, 10 µm.

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