FIG. 2.

Further analysis of selective activation of endosome-associated EGFR by subcellular fractionation and by the time course of immunofluorescence. (A) Subcelllular fractionation. BT20 and MDCK cells were treated with AG-1478, EGF, and monensin as described in Materials and Methods. The cells were then subcellularly fractionated into PM, EN, and CY fractions. The subcellular fractions were subjected to immunoblotting with anti-EGFR, anti-pTyr, and anti-EEA1 antibodies. TL, total lysate. (B) Time course by immunofluorescence of the specific activation of endosome-associated EGFR following wash. BT20 cells were treated with AG-1478, EGF, and monensin for 30 min and then washed with PBS for the indicated times. EGFR (red) and pTyr (green) localization was determined by indirect immunofluorescence. Arrows, colocalization (yellow) of EGFR and pTyr. Bar, 20 μm.