PTP1B activity affects Eph/ephrin-mediated cell segregation. (A) HEK293 and U251 cells were allowed to bind EphA3-Fc and ephrinA5-Fc, respectively, before labeling with Alexa Fluor 647–α-human secondary antibodies and flow cytometry analysis. (B) Cell segregation was monitored in co-cultures of U251 cells and cell tracker green–labeled HEK293 cells in the presence or absence of PTP1B inhibitor (10 µM PTP1B-I), EphA3 inhibitor (10 µM EphA3-I), or DMSO (as control for EphA3-I) for 72 h. Bright-field, fluorescence, and merged images of PFA fixed cell cultures are shown. Contour plots of cell tracker green fluorescent images were generated (MatLab) to illustrate the accumulation of HEK293 cells and the effects of the inhibitor on sorting of mixed HEK293 and U251 cell populations (bottom); their quantification is shown in Fig. S3. (C) Additional quantification of the segregation assay was performed by analyzing ratios of overall fluorescence intensity/area, and the branching ratio of fluorescent areas using ImageJ software. For each setting, cell tracker green images equivalent to 60 fields of view (as shown in B) were analyzed for n = 3 independent experiments. Mean ± SE are shown (error bars; **, P < 0.01; ***, P < 0.001). (D) Co-cultures of HEK293 and U251 cells were treated with PTP1B or EphA3 inhibitor as indicated for 48 h. Lysates and α-EphA3 immunoprecipitates were subjected to Western blot analysis with the appropriate antibodies. Levels of phosphorylated EphA3 relative to total expression were determined by densitometry. Data are representative of at least two independent experiments. Molecular mass standards are indicated next to the gel blots in kilodaltons.