Figure 3.

Hydrogen peroxide (H₂O₂) specifically oxidizes Crk-associated substrate (Cas), focal adhesion kinase (FAK), and Ras-related C3 botulinum toxin substrate 1 (Rac1). A: Biotin-iodoacetamide (BIAM) pull-down analysis of proteins extracted from SK-CO-15 cells after exposure to 1 mmol/L exogenous H₂O₂ for 15 minutes. Protein bands and relative intensity were detected by Western blot analysis using antibodies specific to proline-rich acidic protein 1 (Prap-1), histone deacetylase 4 (HDAC4), FAK, CAS, Abelson murine leukemia viral oncogene homolog (ABL), and Rac1. Blots shown are representative of at least three replicate experiments. B: Immunofluorescence analysis of SK-CO-15 cells for the detection of total FAK and phosphorylated FAK (p-FAK) at residue Y861. Total FAK localized to the cytosol, focal adhesions, nucleus, and cellular junctions, whereas phosphorylated FAK Y861 primarily localizes to cell junctions, as indicated by the white arrowheads. Focal adhesions are indicated by the yellow arrowhead. C: Immunofluorescence analysis of SK-CO-15 cells exposed to 1 mmol/L exogenous H₂O₂ for the detection of phosphorylated FAK at residue Y861, which primarily localizes to cell junctions, as indicated by the white arrowheads. Focal adhesions are indicated by the yellow arrowheads. D: Immunoblot analysis for the detection of phosphorylated FAK at residue Y397 or Y861, and of phosphorylated Cas at residue Y410, Y165, or Y295, before and after exposure of SK-CO-15 to 1 mmol/L H₂O₂. Scale bars = 20 μm (B and C). CTL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.