Repetitive Peroxide Exposure Reveals Pleiotropic Mitogen-Activated Protein Kinase Signaling Mechanisms
Figure 1
Hydrogen peroxide exposure generates increases in tyrosine phosphorylation status and activation of multiple signaling proteins in PC12 cells. (a) Hydrogen peroxide dose-dependent (10–1000 μM) increases in whole-cell protein tyrosine phosphorylation. Hydrogen peroxide exposure time was 10 minutes. Total tyrosine phosphoproteins were purified by immunoprecipitation (IP) from PC12 cells using anti-phosphotyrosine antisera (PY20). Immunoblotting (IB) detection of the phosphotyrosine was achieved with an antisera raised against a differential phosphotyrosine immunogen (PY99). The associated histogram depicts the whole-lane image tyrosine phosphoprotein density quantitation measured as AU-B/px2. (b) Time-dependent hydrogen peroxide (100 μM) increases in whole-cell protein tyrosine phosphorylation. The associated histogram depicts the whole-lane image tyrosine phosphoprotein density quantitation measured as AU-B/px2. (c) Hydrogen peroxide (10 minutes) exposure induces the activation of extracellular signal-regulated kinase (ERK1/2: detected in 2% of the total protein from a whole-cell (w.c.) lysate), tyrosine phosphorylation of Pyk2 and the epidermal growth factor receptor (EGFR) and activation of the non-receptor tyrosine kinase c-Src. (d), (e), (f), and (g) depict the peroxide-induced fold changes in phosphorylation of c-Src, EGFR, Pyk2, and ERK1/2, respectively. Values in each histogram depict the mean ± standard error for three individual experiments for each set of bars. For statistical analysis probability values indicated are *, **, and ***.
