α, ß–unsaturated carbonyls covalently modify cellular PTEN.

(A) 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-PGJ₂ or 4-HNE versus 10 min with 100 µM H₂O₂; 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 H₂O₂. (B) Anti-PTEN immunoblot of MCF-7 cell lysates fractionated by SDS-PAGE under non-reducing conditions. PTEN oxidized to a Cys¹²⁴-Cys⁷¹ disulfide appears as a faster migrating species (PTEN oxidized disulfide) only in cells treated with H₂O₂. This species was undetectable in MCF-7cells treated 30 min with DMSO vehicle or 20 µM each Δ12-PGJ₂, 4-HNE, acrolein or 15-HpETE. (C) Chemical structures of typical α, ß–unsaturated carbonyls. Acrolein and 4-HNE are α, β enals; Δ12PGJ₂ is an α,β enone. Electrophilic β carbons are denoted with δ⁺. Blots are representative of results obtained in at least three independent experiments.