FIG. 4.

Trafficking and activity of EGFR following its selective activation in endosomes. (A) Internalization of inactive EGFR into Rab5-positive endosomes and its subsequent activation. BT20 cells were transfected with wild-type Rab5. Cells were either not treated or treated with EGF for 30 min as controls (left). Some cells were treated with AG-1478 for 15 min, followed by addition of EGF and monensin for 30 min without or with washing and incubation (middle). Some cells were treated with AG-1478 and EGF for 30 min without washing or followed by washing with acidic stripping buffer for 1 min and incubation with medium for 30 min. EGFR (red) and Rab5 (green) localization was determined by indirect immunofluorescence. Arrows, colocalization (yellow) of EGFR and Rab5. (B) Immunoblot analysis of the trafficking and inactivation of EGFR following its selective activation in the endosome. BT20 and MDCK cells were treated with AG-1478 and EGF with or without monensin as described for panel A. The cells were washed and then incubated with serum-free medium for the indicated times. Cell lysates were subjected to immunoblot analysis with mouse anti-pTyr, anti-p-EGFR, and rabbit anti-EGFR antibodies.