Figure 2.

STEP₆₁ binds to and dephosphorylates PTPα at Tyr⁷⁸⁹. (a) The substrate-trapping (C472S) mutants STEP₆₁ C/S and STEP₄₆ C/S or GST tag alone were adsorbed to glutathione sepharose beads and incubated with mouse striatal homogenates. Bound proteins were visualized with specific antibodies as indicated in the figure. (b, c) STEP₆₁ is associated with PTPα in mouse striatum. WT or STEP KO mouse striatal lysates (300 μg) were incubated with mouse IgG and anti-STEP (23E5) mouse monoclonal antibody (b) or goat IgG and anti-PTPα goat polyclonal antibody (c) overnight at 4 °C. Co-immunoprecipitation of STEP interacting proteins (b) or PTPα interacting proteins (c) was probed with antibodies indicated in the figure. Thirty μg of WT striatal lysates were used as input. Representative blots were shown from three independent experiments (n = 3). (d) Recombinant PTPα was phosphorylated by Fyn in vitro. (e) STEP₆₁ dephosphorylates PTPα at Tyr⁷⁸⁹. In vitro phosphorylated recombinant PTPα was incubated with active STEP₆₁ (WT) or inactive STEP₆₁ (C/S). The residual phosphorylation of PTPα at Y⁷⁸⁹ was assessed using a phospho-specific antibody. Data were expressed as mean ± SEM (WT versus C/S at the same dose: **p < 0.01, one-way ANOVA with post hoc Tukey’s test; n = 4).