hours after transfection. COS-7 cells were incubated with EGF (0.1 g/ml) [biotinylated, complexed to Texas Red-streptoavidin (Molecular Probes, Eugene, OR)] in binding buffer [20 mM Hepes-NaOH ( pH 7.5), 130 mM NaCl, and 0.1% bovine serum albumin] at 4°C for 60 min. Internalization of EGF was allowed by incubation in Dulbecco's modified Eagle's medium at 37°C for 10 min, then excess EGF was removed with 0.2 M AcOH ( pH 2.5) and 0.5 M NaCl at 4°C for 5 min. Cells were fixed in 3.7% formaldehyde, permeabilized with 0.2% Triton X-100, and immunostained with a polyclonal antibody to myc (Santa Cruz Biotechnology, Santa Cruz, CA) and fluorescein isothiocyanate-conjugated antibody to rabbit (Organon Teknika, Boxtel, Netherlands). Internalization of EGF was observed by confocal microscopy (Bio-Rad). 37. We thank Y. Watanabe (Ehime University, Japan) for providing us with various synthetic phosphoinositides.

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