FIGURE 5.

Kinase assays of PtpA dephosphorylation of GSK3α in vitro. A, dephosphorylation of GSK3α by PtpA was tested in an in vitro kinase assay. GSK3α (1 and 2 μm) was autophosphorylated in a kinase buffer containing 10 μCi of [γ-³²P]ATP with or without PtpA (0.04 μm) and was resolved onto a 12% SDS gel and exposed to a PhosphorImager screen for radiolabeled band localization. After drying the gel, bands corresponding to phosphorylated GSK3α were cut from the gel, and the radioactive incorporation was measured by a scintillation counter. This graph represents the radioactivity of the dried gel. Results are expressed as ± S.D. of three independent experiments. The p value of 2 μm GSK3α with PtpA is 0.0214. B and C, inhibiting effect of PtpA on GSK3α's activity was tested by radiometric analysis. GSK3α (2–4 μm) was autophosphorylated in a reaction containing kinase buffer and 10 μCi of [γ-³²P]ATP. PtpA (0.04 μm) and Na₃VO₄ (1.5 mm) or PtpA (0.04 μm) and BVT 948 (5 μm) were added to the sample mixtures and were spotted onto phosphocellulose paper. Radioactivity levels were measured by a scintillation apparatus. *, p < 0.05; **, p < 0.001; ***, p < 0.0001. Significant difference is compared by Student's t test.