FIG.1.

Analysis by immunofluorescence of selective activation of EGFR after its endocytosis into endosomes. (A and B) BT20 cells were treated with AG-1478 for 15 min and then stimulated with EGF in the presence of monensin at 37°C for 30 min. Some of the cells were then washed three times with PBS and incubated with serum-free medium for 30 min. BT20 cells that were serum starved or treated only with EGF for 15 and 30 min at 37°C were used as controls; EGFR (red) and pTyr (green) (A) or p-EGFR (green) (B) localization was determined by indirect immunofluorescence. Arrows, colocalization (yellow) of EGFR and pTyr (A) or EGFR and p-EGFR (B). (C) BT20 cells were treated with AG-1478 for 15 min and then stimulated with TR-EGF in the presence of monensin at 4°C for 30 min followed by incubation at 37°C for the indicated times. Some of the cells were then washed with PBS three times and incubated with serum-free medium for the indicated times. BT20 cells that were treated only with TR-EGF at 4°C for 30 min and then at 37°C for indicated times were used as controls; TR-EGF (red), EGFR (green), and pTyr (blue) localization was determined by triple indirect immunofluorescence as described in Materials and Methods. Arrows, colocalization (yellow) of TR-EGF and EGFR; arrowheads, colocalization (light purple) of TR-EGF, EGFR, and pTyr. (D) MDCK cells were treated with AG-1478 for 15 min and then stimulated with EGF in the presence of monensin at 37°C for 30 min. Some of the cells were then washed with PBS three times and incubated with serum-free medium for 30 min. MDCK cells that were serum starved or treated only with EGF for 15 and 30 min at 37°C were used as controls; EGFR (red) and pTyr (green) localization was determined by indirect immunofluorescence. Arrows, colocalization (yellow) of EGFR and pTyr. Bars, 20 μm.