Resveratrol Inhibit LPS-Induced Activation by mTOR Figure 10. Inhibition of phosphorylation of IkB-a, CREB, and MAPKs signaling by resveratrol is mTOR-dependent during BV-2 cells activation by LPS. Approximately 16106 cells/ml were seeded in six-well plates and incubated until 80% confluency. Cells were pre-treated with resveratrol (25, 50, and 100 mM) for 1 h in the absence or presence of rapamycin (10 nM), then exposed to LPS (1 mg/ml) for 30 min. Cell lysates (50 mg protein) were prepared and subjected to Western blot analysis by using antibodies specific for phosphorylated forms of IkB-a, CREB, ERK1/2, JNK and p38 MAPK (shown as phospho-IkB-a, etc.) as described in the methods. Equivalent loading of cell lysates was determined by reprobing the blots with anti-b-actin, total ERK1/2, JNK or p38 MAPK antibodies. The relative protein levels were quantified by scanning densitometry and normalized to b-actin, total ERK1/2, JNK or p38 MAPK. The values shown are mean 6 SEM of data from three independent experiments. #Significant compared with control alone, p,0.05. *Significant compared with LPS alone, p,0.05. nSignificant compared with resveratrol+LPS, p,0.05. doi:10.1371/journal.pone.0032195.g010 PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e32195