Fig. 1. Nonspecific catalytic activity of PTPα from unsynchronized and mitotic cells.

Approximately equal amounts of overexpressed human WT PTPα-HA, PTPα(S180A/S204A)-HA, PTPα(S180A)-HA, and PTPα(S204A)-HA were immunoprecipitated with anti-HA antibody from lysates from NIH3T3-derived overexpresser cells that were either unsynchronized (U) or arrested in mitosis (M). Aliquots of immunoprecipitates that had been washed with a high salt buffer were incubated with [³²P]Tyr(P)-containing MBP in phosphatase buffer for 30 min at 30 °C or subjected to anti-Tyr(P) or anti-PTPα immunoblotting. Additional experiments were performed similarly, except that the WT PTPα and PTPα(S180A/S204A) immunoprecipitates were preincubated with PP2A, a Ser/Thr phosphatase, and then washed with phosphatase buffer before incubation with MBP. A, shown is the amount of ³²P released per molecule of PTPα after a 30-min incubation normalized to the amount released by overexpressed PTPα from unsynchronized cells. Error bars indicate the S.E.M. from four to five experiments. B, shown is the anti-Tyr(P) (Anti-pTyr) immunoblot of the immunoprecipitated PTPα. C, shown is the anti-PTPα immunoblot of the immunoprecipitated PTPα. D, experiments were performed as described above using immunoprecipitated WT PTPα-HA or PTPα(S180A/S204A)-HA, except that the immunoprecipitates were preincubated with (+) or without (−) PP2A prior to PTP assay with ³²P-labeled MBP. The amount of ³²P released per molecule of PTPα after a 30-min incubation normalized to the amount released by PP2A-untreated PTPα from unsynchronized cells is shown. Error bars indicate the S.E.M. from two to three experiments. E, shown is the anti-Tyr(P) immunoblot of the immunoprecipitated PTPα. F, shown is the anti-PTPα immunoblot of the immunoprecipitated PTPα. SDS-PAGE was performed on 10% gels. The positions of molecular mass standards are indicated in kilodaltons.