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Effects of MAP kinase inhibitors on H<sub>2</sub>O<sub>2</sub>-promoted eNOS activation and phosphorylation.

By Juliano L. Sartoretto (138024), Hermann Kalwa (138027), Takashi Shiroto (138030), Simone M. Sartoretto (138033), Michael D. Pluth (138037), Stephen J. Lippard (138040) and Thomas Michel (138043)

Abstract

<p>Panel A shows the results of immunoblots analyzed in lysates prepared from adult murine cardiac myocytes treated with hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>, 25 µM) for the indicated times. Cell lysates were analyzed in immunoblots probed using antibodies directed against phospho-MEK (Ser217/221), phospho-ERK1/2 (Thr202/Tyr204), total MEK, ERK, and GAPDH, as indicated. Below each immunoblot are the results of densitometric analyses from pooled data, showing the fold increase in protein phosphorylations (in arbitrary units) in cardiac myocytes treated with H<sub>2</sub>O<sub>2</sub> at the indicated times plotted relative to the signals present in unstimulated cells. Each data point represents the mean ± SE derived from three independent experiments. The results are significant at the p<0.05 level. *indicates p<0.05 (<i>ANOVA</i>). Panel B adult mouse cardiac myocytes were loaded with the NO dye Cu<sub>2</sub>(FL2E), and then treated with PD98059 (37.4 µM), MEK inhibitor (1 µM) or vehicle followed by hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>, 10 µM) treatment. Upper panel shows representative fluorescence images at 0, 2, 5, and 10 minutes followed treatments as indicated. Lower panel shows representative fluorescence tracings of a cell treated with PBS (green line), H<sub>2</sub>O<sub>2</sub> (red line), H<sub>2</sub>O<sub>2</sub> in the presence of MEK1/2 inhibitor (purple line), or H<sub>2</sub>O<sub>2</sub> in the presence of PD98059 (blue line). The results shown are representative of three independent experiments that yielded equivalent results. In panel C, cardiac myocytes were incubated with PD98059 (50 µM, 30 min) or vehicle, then treated with H<sub>2</sub>O<sub>2</sub> (25 µM, 15 min) and analyzed in immunoblots probed with antibodies as shown. Panel D shows immunoblot analyses from cardiac myocytes incubated with MEK inhibitor (1 µM, 30 min) or vehicle, then treated with H<sub>2</sub>O<sub>2</sub> (25 µM, 15 min). Below each representative immunoblot are shown the results of densitometric analyses from pooled data, documenting the changes in phospho-eNOS (Ser1177) plotted relative to the signal present in unstimulated cells. Each data point represents the mean ± S.E. derived from at least three independent experiments; *indicates p<0.05 (<i>ANOVA</i>).</p

Topics: Biochemistry, Cell Biology, Neuroscience, kinase, inhibitors, enos, activation
Year: 2012
DOI identifier: 10.1371/journal.pone.0044627.g005
OAI identifier: oai:figshare.com:article/252184
Provided by: FigShare
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