Fig. 2.

Ten14 exerts its effects mainly by the compartmentalization of EGFR at the cell surface. NR6WT cells were quiesced, and then stimulated with indicated doses of EGF for the set time points or 5 min at 37°C and 4°C.Cells were then lysed, proteins separated through SDS–PAGE gels, and immunoblotted for the indicated proteins. Shown are representative blots of at least four for each analysis. For immunofluorescence studies, NR6WT cells were treated with EGF at 4°C and assayed for EGFR localization. A: Over a 5 min period, EGF leads to robust dose-dependent activation of ERK/MAPK at 37°C, accompanied by phosphorylation of PLCγ1 and SHC. However, at 4°C, the activation profile for EGF-treated cells changes, with increased phosphorylation of PLCγ1 and SHC and very low levels of ERK/MAPK activation. This profile is strikingly is similar to that observed with Ten14 at 37°C (see Fig. 1B). B: A differential activation profile is observed for EGF-treated cells over a period of 30 min, where the robust activation of ERK/MAPK seen at 37°C is lost at 4°C, but increased phosphorylation of PLCγ1 is observed. C,D: For cells treated with EGF at 4°C, active EGFR is localized predominantly at the cell surface, suggesting that the effects observed with Ten14 at 37°C and EGF at 4°C (right part) is driven mainly through compartmentalization of active EGFR at the plasma membrane.