Abrogated PTP1B expression causes enhanced and prolonged EphA3 activation. (A and B) EphA3/HEK293Ts (A) and 22Rv1 cells (B), stably transfected with PTP1B-shRNA– or nontarget control vector–containing lentiviral transduction particles, were subjected to ephrinA5 stimulation. α-EphA3 immunoprecipitates and whole cell lysates were examined by immunoblotting with the appropriate antibodies. Densitometry quantifies EphA3 phosphorylation relative to total EphA3 expression. Data are representative of at least five (A) and three (B) independent experiments, respectively. (C) The time course of ephrinA5-induced EphA3 phosphorylation in PTP1B-reconstituted or parental PTP1B^−/− MEFs, both stably transfected with EphA3, was assessed by immunoblotting of anti-EphA3 immunoprecipitates using antibodies against EphA3, PY, and PTP1B, as indicated. Densitometry quantifies relative EphA3 phosphorylation, which is representative of two experimental repeats. The black line indicates that intervening lanes have been spliced out. (D) PTP1B^−/− MEFs with or without reconstitution with w/t PTP1B and w/t MEFs were transiently transfected with EphA3. α-EphA3 immunoprecipitates from ephrinA5-stimulated or nonstimulated cells were analyzed by Western blot analysis using antibodies against PY-EphA3, EphA3, and PTP1B as indicated. Molecular mass standards are indicated next to the gel blots in kilodaltons.