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. 2017 Sep 8;7:10981. doi: 10.1038/s41598-017-11496-3

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Characterization of macrophage eicosanoid catabolism enzyme mRNA expression and 5-LO protein synthesis. For mRNA expression (A) BMDMs were treated with IFN-γ (100 ng/mL) (M1), IL-4 + IL-13 (10 ng/mL) (M2), or only adhered (M0) for 2, 6, and 24 h in vitro; or (B) resting PMs were used. Total RNA was extracted, synthesized as cDNA, and the relative expression (ΔΔCt) was analysed by qRT-PCR. Transcripts encoding cyclooxygenase and lipoxygenase pathway genes were analysed. The results were normalized to endogenous expression of the internal controls Actb and Gapdh. The blue dotted lines show the mRNA expression on non-primed BMDMs (M0). The results are presented as the means ± s.e.m. of three independent experiments (n = 3). *p < 0.05 compared to non-primed BMDMs (M0). Qualitative data for (C) 5-LO protein synthesis was demonstrated by Western Blot assay using a rabbit-polyclonal anti-5-LO antibody (78 kDa). The membranes were incubated with specific conjugated secondary antibodies (goat anti-rabbit IgG-HRP) and detected with chemiluminescence (ECL) reagent. Representative result from two independent experiments is shown.