Figure 6.

pY374-PKCδ and PTPN14 are sequentially recruited to EGFR-containing endosomes after EGF stimulation. Cell surface EGFR (SUM149PT cells) was labeled with EGFR-ECD antibody before inducing endocytosis with EGF (20 ng/ml). After fixation, EGFR was labeled with Alexa Fluor 594 while all other proteins shown were labeled with Alexa Fluor 488 at the indicated times after fixation. (a–c) Time course of colocalization of EGFR-containing endosomes (EGFR, Alexa Fluor 594, false-colored green) with pY374-PKCδ or PTPN14 (Alexa Fluor 488, false-colored red) as indicated. Representative low- and high-magnification and split-color confocal micrographs of EGFR-containing endosomes costained with either pY374-PKCδ (a) or PTPN14 (b) as indicated at the indicated times after initiating EGFR trafficking (arrowheads indicate colocalization), and time course of colocalization of EGFR-containing endosomes with either pY374-PKCδ or PTPN14 quantified from confocal micrographs (c; mean ± SEM; n > 30 cells per time point).