FIG. 6.

Stimulation of various signal transduction pathways by activation of endosome-associated EGFR. BT20 and MDCK cells were treated with AG-1478 and EGF with or without monensin as described in Materials and Methods. The cells were washed and then incubated with serum-free medium for the indicated times. (A) Activation of Ras. BT20 cell lysates were incubated with GST-RBD conjugated with glutathione beads. The glutathione beads were then subjected to immunoblot analysis with a mouse anti-Ras antibody. GST-Raf RBD was stained by a mouse anti-GST antibody to show equal loading. (B) Activation of ERK. BT20 and MDCK cell lysates were subjected to immunoblot analysis with a rabbit anti-phospho-ERK1/2 (p-ERK1/2) antibody. Total ERK1/2 was used as the control. (C) Activation of Akt and its inhibition by wortmannin. Activation of Akt was determined by an anti-phospho-Akt (p-Akt) antibody. To determine the effects of wortmannin on the activation of Akt, BT20 and MDCK cells were treated with wortmannin (100 nM) for 15 min before the treatment with AG-1478, EGF, and monensin. Cell lysates were subjected to immunoblot analysis with an anti-p-Akt antibody. (D) Activation of PLC-γ1. Activation of PLC-γ1 was determined by immunoblotting of BT20 cell lysates or EGFR immunoprecipitates (IP) with anti-phospho-PLC-γ1 (p-PLC-γ1) antibody.