PTEN is oxidized and inhibited in response to fMLP. (A) Mouse neutrophils were preincubated with Trolox (100μM) or mock-treated for 10 minutes before exposure to 1μM fMLP or 1mM H₂O₂ for 1 minute. Lysates were analyzed by SDS-PAGE under nonreducing conditions and immunoblotted for PTEN to detect oxidized and reduced forms of PTEN. fMLP-induced oxidation of PTEN was prevented by Trolox. Treatment of lysate with DTT was used as a positive control for the reduced form. Densitometry was used to quantify the ratio between oxidized and reduced PTEN (n = 3) and the averages ± SD are shown. (B) Partially permeabilized neutrophils were preincubated with Trolox (100μM) or mock-treated for 10 minutes before stimulation with 1μM fMLP. A PTEN-enzymatic activity assay was performed with the soluble PTEN substrate diC8 for 30 minutes. P_i-release was quantified using a Malachite Green assay, and the means ± SD of 3 independent experiments are shown.