Elevated expression of w/t PTP1B or PTP1B-[D-A] inhibits EphA3 endocytosis. (A) EphrinA5-Fc stimulated (10 min) EphA3/HEK293T cells expressing exogenous GFP-PTP1B-[D-A] or w/t GFP-PTP1B were fixed, permeabilized, and labeled with α-EphA3 and Alexa Fluor 546–labeled secondary antibodies for confocal microscopy. Individual fluorescent channels and merged images are shown. Closed and open arrowheads denote cells with highly elevated and low levels of PTP1B, respectively. Areas of interest are boxed and shown at increased magnification (insets). (B) The level of biotinylated EphA3 on the surface of AP_N-EphA3/TM-BirA/HEK293T cells was determined by flow cytometry using Alexa Fluor 594–monoSA. The Alexa Fluor 594–monoSA intensity of ephrin-stimulated (20 min) cells is reduced compared with untreated cells, which reflects EphA3 internalization (↔). (C) The relative ephrinA5-induced change in cell surface EphA3 was determined as in B in cells transfected with pEGFP (control), w/t GFP-PTP1B, or GFP-PTP1B–[D-A] at cDNA concentrations of 0.15, 0.3, or 0.6 µg/well as indicated. Mean ± SE from three individual samples from each condition are shown (error bars). (D) 22Rv1 cells transfected with EGFP, GFP-PTP1B-[D-A], or w/t GFP-PTP1B were stimulated with ephrinA5-Fc for 20 min, and cell surface EphA3 was labeled using sheep α-EphA3 and Alexa Fluor 647 secondary antibodies, analyzed by flow cytometry, and evaluated as described in B. Mean ± SE from six individual samples of two separate experiments are shown (error bars; *, P < 0.05; ***, P < 0.001).