FIGURE 3.

DMS relieves the inhibitory action of ANP32A on PP2A. A, HUVEC were treated with 10 μm DMS or DHS for various times, and PP2A activity was determined as described under “Experimental Procedures.” PP2A was immunoprecipitated, and PP2A immunoblots are represented in the inset. Data represent mean ± S.D. of at least two independent experiments performed in duplicate. B, HUVEC were treated with ANP32A siRNA or control siRNA for 48 h. ANP32A and GAPDH protein levels were measured by immunoblot analysis. C, HUVEC were treated with ANP32A siRNA (80 nm) or control siRNA (80 nm) for 48 h, followed by treatment with DMS (10 μm) for 2 h or not and PP2A activity was determined. PP2A was immunoprecipitated, and PP2A immunoblots are represented in the inset. Data represent mean ± S.D. of at least two independent experiments performed in duplicate. D, PP2A dimer (containing A and C subunits) was incubated with increasing concentrations of ANP32A (0–120 nm), and its effects on PP2A activity was determined in vitro using the radioactive phosphatase assay, as described under “Experimental Procedures.” Data represent mean ± S.E. of at least three independent experiments performed in duplicate n = 6. E, purified ANP32A (40 nm) was incubated with various concentrations of DMS (0, 0.25, or 1.25 μm), and PP2A dimer activity were determined in vitro as described above. The data represent the mean ± S.E. of at least four independent experiments performed in duplicate n = 8.