MPTAG prevents the TNF-α-induced NF-κB transcription and activation in human endothelial cells.

<p>(A) The cells were pretreated with or without 400 µM of MPG before induction with TNF-α (10 ng/ml) for 4 h. The total RNA of the cells was isolated and analysed by RT-PCR. The intensity of transcripts was normalized with that of β-Actin levels expressed under similar conditions. (B) The cells were pretreated with MPTAG at varying concentrations and then induced with TNF-α. The cytoplasmic (CE) and nuclear (NE) extracts were prepared from untreated and MPTAG-treated TNF-α-stimulated cells (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046528#s4" target="_blank">Methods</a>”). The nuclear extracts were analyzed for NF-κB activation by EMSA. (C) The nuclear extracts from unstimulated or TNF-α-stimulated HUVECs were incubated with the indicated antibodies and analyzed for NF-κB activation by EMSA (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046528#s4" target="_blank">Methods</a>”). (D) The cells were transiently transfected by electroporation with a NF-κB-containing luciferase reporter gene followed by treatment with 400 µM MPTAG and TNF-α stimulation. The supernatants after cell lysis were assayed for luciferase activity. The mean value for cells treated with no MPTAG and no TNF-α was set to 1, and -fold differences were determined by comparing values against this set value. *p<0.005 vs. uninduced cells; **p<0.01 vs. TNF-α-induced cells, statistical difference was set at p<0.05.