Journal: Nature Communications
Article Title: Opposing roles of LTB 4 and PGE 2 in regulating the inflammasome-dependent scorpion venom-induced mortality
doi: 10.1038/ncomms10760
Figure Lengend Snippet: ( a ) Macrophages were pre-treated with LTB 4 (0.01–100 nM, 10 min), before TsV (50 μg ml −1 ) addition for 24 h. Supernatants were collected for IL-1β quantification. ( b ) J774.1 macrophages were pre-treated or not with BLT1 antagonist (U-75302, 1 μM, 30 min) and with LTB 4 (100 nM, 10 min) or not before TsV (50 μg ml −1 ) addition for 24 h. Supernatants were collected for IL-1β quantification. In another set of experiments, ( c ) J774.1 macrophages were transfected for 48 h to specifically silence Ltb4r1 and Ltbr4r2 gene expression. Cells were then pre-treated or not with LTB 4 (100 nM, 10 min), before TsV (50 μg ml −1 ) addition for 6 h. In other experiment, ( d ) Ltb4r1 and Ltb4r2 mRNA expression in C57BL/6 peritoneal macrophages was determined following TsV stimulation (50 μg ml −1 ) at 2 and 6 h by qRT–PCR. ( e ) cAMP was determined following stimulation with TsV (50 μg ml −1 , 5 min) or PGE 2 (1 μM, 3 min); or LTB 4 (10 nM, 1 min) followed by TsV or PGE 2 . ( a – e ) Data are representative of two independent experiments performed in quadruplicate. Significant differences P <0.05 are marked with symbols and the error bars denote s.d. *Medium only versus stimulus or treatments, and # TsV versus treatments, according to one-way ANOVA with Bonferroni's post-test.
Article Snippet: J774.1 macrophages (1 × 10 5 ) were seeded at 60–70% confluence and transfected with 60 pmol of Ltb4r1 (sc-42587) and Ltb4r2 (sc-45323) siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using Lipofectamine as the transfection reagent (Life Technologies, Carlsbad, CA, USA) and Opti-MEM reduced serum medium for culture (Life Technologies).
Techniques: Transfection, Expressing, Quantitative RT-PCR
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