<p>(<b>A</b>) Diagram of the procedure to identify PTEN with an oxidized or alkylated thiol in cells exposed to ROS or α, ß–unsaturated carbonyls. The anti-PTEN immunoblot shows oxidized or carbonylated PTEN (NA Pulldown) relative to total PTEN (Cell Lysate) isolated from MCF-7 cells treated 30 min with vehicle (DMSO), 10 µM Δ12-PGJ<sub>2</sub> or 4-HNE versus 10 min with 100 µM H<sub>2</sub>O<sub>2</sub>; an immunoblot from a separate experiment shows oxidized, carbonylated and total PTEN in cells treated for 30 min with vehicle or 20 µM acrolein versus 10 min with 100 µM H<sub>2</sub>O<sub>2</sub>. (<b>B</b>) Anti-PTEN immunoblot of MCF-7 cell lysates fractionated by SDS-PAGE under non-reducing conditions. PTEN oxidized to a Cys<sup>124</sup>-Cys<sup>71</sup> disulfide appears as a faster migrating species (PTEN oxidized disulfide) only in cells treated with H<sub>2</sub>O<sub>2</sub>. This species was undetectable in MCF-7cells treated 30 min with DMSO vehicle or 20 µM each Δ12-PGJ<sub>2</sub>, 4-HNE, acrolein or 15-HpETE. (<b>C</b>) Chemical structures of typical α, ß–unsaturated carbonyls. Acrolein and 4-HNE are α, β enals; Δ12PGJ<sub>2</sub> is an α,β enone. Electrophilic β carbons are denoted with δ<sup>+</sup>. Blots are representative of results obtained in at least three independent experiments.</p
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