Inhibition of the phosphorylation of CREB, and MAPKs signaling by resveratrol is PI3-K-dependent during RAW 264.7 macrophage cells activation by LPS.

<p>Approximately 1×10⁶ cells/ml were seeded in six-well plates and incubated until 80% confluency. Cells were pre-treated with resveratrol (1, 5, and 10 µM) for 1 h in the absence or presence of wortmannin (1 µM), then exposed to LPS (5 µg/ml) for 30 min. Cell lysates (50 µg protein) were prepared and subjected to Western blot analysis by using antibodies specific for phosphorylated forms of CREB, ERK1/2, JNK and p38 MAPK (shown as phospho-IκB-α, etc.) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044107#s4" target="_blank">methods</a>. Equivalent loading of cell lysates was determined by reprobing the blots with anti-β-actin, total ERK1/2, JNK or p38 MAPK antibodies. The relative protein levels were quantified by scanning densitometry and normalized to β-actin, total ERK1/2, JNK or p38 MAPK. The values shown are mean ± SEM of data from three independent experiments. ^#Significant compared with control alone, <i>p</i><0.05. ^*Significant compared with LPS alone, <i>p</i><0.05. ^ΔSignificant compared with resveratrol + LPS, <i>p</i><0.05.