FIGURE 4.

A, Western blot showing PTEN expressed under control conditions (con) in guinea pig ventricular myocytes (gp-m) and following incubation with 10 μm As₂O₃ for 4 h. Co-incubation of 10 μm As₂O₃ together with 10 mm NAC prevented oxidation of PTEN. PTEN was immunoprecipitated from whole cell lysates prior to detection on non-reducing SDS-PAGE. Arrowhead, position of oxidized PTEN. B, quantitative analysis of PTEN oxidation in cardiomyocytes on exposure to As₂O₃ (n = 3–4). As₂O₃ increases the oxidized form of PTEN significantly (Dunnett's test, p < 0.05). C, detection of oxidized PTEN by phosphatase assay. Cardiomyocytes were incubated for 4 h either under control conditions or in the presence of 10 μm As₂O₃ and lysed in the presence of iodoacetamide. PTEN was immunoprecipitated and assayed for phosphatase activity under reducing conditions. Measurements are expressed as the amount of phosphate released from PIP₃ by PTEN. Note, that phosphate release is different between control- and As₂O₃-treated myocytes at the p < 0.05 level. D, detection of total PTEN activity by phosphatase assay. Cardiomyocytes were incubated for 4 h either under control conditions or in the presence of 10 μm As₂O₃ and lysed under reducing conditions. Importantly, total phosphate release was not different between control and As₂O₃-treated myocytes. IP, immunoprecipitation; WB, Western blot. Error bars, S.E.