FIG. 3.

Selective activation of endosome-associated EGFR without monensin. (A) BT20 cells were treated with AG-1478 for 15 min and then stimulated with TR-EGF at 4°C for 30 min, followed by a wash with acidic stripping buffer at 4°C for 1 min. The cells were then washed with PBS three times and incubated with medium at 37°C for the indicated times. BT20 cells were triple fluorescence stained for TR-EGF (red), EGFR (green), and p-EGFR (blue) as described in Materials and Methods. Arrows, colocalization (yellow) of TR-EGF and EGFR; arrowheads, colocalization (light purple) of TR-EGF, EGFR, and p-EGFR. Bar, 20 μm. (B) Subcelllular fractionation. BT20 and MDCK cells were treated with AG-1478 for 15 min and then stimulated with EGF at 37°C for 30 min, followed by a wash with acidic stripping buffer at 4°C for 1 min. The cells were then washed with PBS three times and incubated with medium at 37°C for the indicated times. The cells were then subcellularly fractionated into the PM, EN, and CY fractions. The subcellular fractions were subjected to immunoblotting with anti-EGFR, anti-pTyr, and anti-EEA1 antibodies as described in Materials and Methods. TL, total lysate.