FIGURE 2.

ErbB2-mediated H₂O₂ production enhanced phospho-tyrosine signaling and PTPα-reversible oxidation in 10A. B2 cells. A, ErbB2-induced H₂O₂ production was assessed by molecular imaging using PF6-AM. Serum-starved 10A.B2 cells were loaded with 5 μm PF6-AM for 20 min and stimulated with 1 μm AP1510 for the indicated times (t) (in minutes) and then imaged. Alternatively, 100 μm H₂O₂ was added to the medium for 5 min. For diphenyleneiodonium (DPI) treatment, cells were preincubated in medium containing 10 μm DPI for 30 min prior to AP1510 stimulation for 5 min. Scale bars = 50 μm. B, DPI inhibition of ErbB2-induced tyrosine phosphorylation. Serum-starved 10A.B2 cells were stimulated with 1 μm AP1510 in the presence or absence of DPI (10 μm, 30 min) for the indicated times. Tyrosine-phosphorylated proteins were immunoprecipitated (IP) from 200 μg of cell lysate using PT-66 and immunoblotted using 4G10 anti-pTyr antibodies. ErbB2 was detected using anti-HA antibodies. Loading controls were performed by immunoblotting lysates for actin. C, schematic of the cysteinyl labeling assay (31). IAA, iodoacetic acid; IAP, iodoacetyl-PEG2. D, serum-deprived 10A.B2 cells were incubated with AP1510 (1 μm) for the indicated times and subjected to the cysteinyl labeling assay. Biotinylated proteins were purified on streptavidin-Sepharose (s-S) beads, resolved on SDS-PAGE, and immunoblotted for PTPα. Lysates were also probed for PTPα as controls. PD, pull down.