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. 2012 May 15;52(10):2082–2090. doi: 10.1016/j.freeradbiomed.2012.03.022

Fig. 4.

Fig. 4

Ascorbate leads to increased eNOS phosphorylation via AMPK activation. (A) EA.hy926 cells were pretreated with 20 μM compound C for 30 min and then incubated with 100 μM ascorbate for 1 h. Western blot and subsequent densitometric analyses were performed to detect and quantify (phospho-) eNOS protein levels. One representative blot is shown. Band intensities were normalized to tubulin and expressed as fold untreated control (⁎p<0.05; ns, not significant; mean±SEM, n=3). (B) HUVECs were transfected with AMPKα siRNA or scrambled control before treatment with 100 μM ascorbate for 1 h as indicated. Western blot and subsequent densitometric analyses were performed to detect and quantify (phospho-) eNOS and AMPK protein. One representative blot is shown. Band intensities were normalized to actin and expressed as fold untreated control (⁎p<0.05; ns, not significant; mean±SEM, n=4). (C) EA.hy926 cells or (D) HUVECs were treated with 100 μM ascorbate for 5 min to 1 h. Western blot and subsequent densitometric analyses were performed to detect and quantify (phospho-) AMPK protein levels. One representative blot is shown. Band intensities were normalized to tubulin and expressed as fold untreated control (⁎p<0.05; ⁎⁎p<0.01; ns, not significant; mean±SEM, n=3). (E) EA.hy926 cells were treated for 1 h with the indicated concentrations of ascorbate and subjected to Western blot analysis for the detection of (phospho-) AMPK levels. One representative blot is shown. Band intensities were normalized to actin and expressed as fold untreated control (⁎⁎⁎p<0.001; ns, not significant; mean±SEM, n=4).