MAPKs activation and toxin-induced COX-2, TNF-α, IL-8 and IκBα mRNA expression upon p38 MAPK and JNK inhibition.

<p>(<b>A</b>) Western blot analysis of MAPK phosphorylation in macrophages incubated 4 h with the <i>O</i>. cf. <i>ovata</i> toxin extract (final OSTRTX concentration 2 ng/ml and 20 ng/ml) (lane 2, 3), 2 ng/ml PLTX (lane 4) or the vehicle (lane 1). 20 µg of whole cell extracts were separated by SDS-PAGE on 8% gel and submitted to western immunoblot with phospho-specific antibodies against ERK1/2, JNK and p38 MAPK. Actin was stained as a loading control. (<b>B</b>) Total RNA was extracted from macrophages pre-incubated 1 h with 1 µM p38 inhibitor (ip38) or 1 µM and 5 µM JNK inhibitor (iJNK), and then exposed 4 h to 2 ng/ml palytoxin. In parallel, macrophages were left untreated and then exposed to PLTX or to the vehicle alone. RNA was amplified with gene-specific primers. Expression data, normalized to the housekeeping B2M gene, were analyzed by the 2^−ΔΔCT method and referred to the value obtained in control cells (CTR). The boxplots show the results of 3 independent experiments, run in duplicate. Asterisks indicate statistical significance versus cells receiving only PLTX (*<i>p</i><0.05; **<i>p</i><0.01).