(A) ROS oxidize thiols in redox-sensitive phosphatases (PTPs). Maleimide groups react efficiently and specifically with reduced sulfhydryls (R-S^–) of PTPs to form stable thioester bonds but do not react and bind R-SOH oxidized sulfenic acid (thioester) forms. (B) Cell were treated with vehicle, H₂O₂ (15 μM), or Ac2-26 (3 μM) for 15 minutes and subjected to labeling with biotin-NM. Biotinylated proteins were precipitated with SA beads and subjected to immunoblot analysis using PTEN, PTP-PEST, and SHP2 antibodies. β-Actin was used as a loading control. Immunoblots presented are representative of 3 independent experiments. Normalized signal intensity is indicated above the blots. (C) SK-CO15 cells were incubated for 30 minutes at room temperature with 100 μM GTPγS, vehicle, or Ac2-26 peptide for 15 minutes. Active Rac1 (Rac1-GTP) was pulled down using agarose bead–conjugated GST-PAK-RBD. Total (Rac1) and active Rac1 (Rac1-GTP) were analyzed by immunoblot. Immunoblots are representative of 3 independent experiments. (D) Active Rac1 pull-down samples were subjected to immunoblot analysis with antibodies against Vav2, p120, and NOX1. Immunoblots are representative of 3 independent experiments. (E) ROS generation was analyzed in SK-CO15 cells, treated with hydro-Cy3 dye, by confocal microscopy. Cells were incubated with vehicle, Ac2-26 (3 μM), or Ac2-26 plus NSC23766. Micrographs are representative of 3 independent experiments. Scale bar: 20 μm. (F) SK-CO15 cells were transfected with siRNA targeting p120 catenin. Three days after siRNA transfection, cells were treated with Ac2-26 for 15 minutes, and ROS generation was detected. Confocal micrographs are representative of 4 independent experiments. Scale bar: 20 μm. Summarized data for hydro-Cy3 fluorescence intensity are presented in the graphs (mean ± SEM,*P < 0.05 vs control, ^#P < 0.05 vs. Ac2-26, n = 3).