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2 protocols using arachidonyl trifluoromethyl ketone

1

Mouse cPLA2α Gene Silencing Protocol

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If not otherwise stated, reagents were supplied by Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Arachidonyl trifluoromethyl ketone (ATK), an effective inhibitor of cPLA2, was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). The siRNAs targeting the mouse cPLA2α gene and the sequences of RNAi were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Three gene sequences were selected for RNAi, which were verified by experiments to suppress the expression of mouse cPLA2α in L929 cells (18 (link)). The siRNA sequences (sense strand) were as follows: No. 1, 5′-GCGAACGAGACCTTCAAT-3′; no. 2, 5′-GGTGCATAACTTCATGCTG-3′; no. 3, 5′-GCACATCGTGAGTAATGAC-3′. 5′-TTCTCCGAACGTGTCACGT-3′ served as a negative control. A BLAST search on the NCBI website was used to confirm that the sequences did not have homology to any other mouse genes.
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2

Mast Cell Degranulation Pathway Inhibitors

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OVA, DNP-IgE, PIPES, 4-nitrophenyl-N-acetyl-β-D-glucosamide, BAY61-3606, Rottlerin, and BAY11-7082 were purchased from Sigma-Aldrich (MO, USA). Arachidonyl trifluoromethyl ketone (ATK) was from Cayman Chemical (MI, USA). Dexamethasone sodium phosphate (Dex) was purchased from Rongsheng Pharmaceutical Co., Ltd. (Jiaozuo, China). Aluminum hydroxide gel was from Meihua chemical industry limited company (Shanghai, China). All enzyme-linked immunosorbent assay (ELISA) kits were from BioLegend (CA, USA). ABC kit was obtained from Zhongshan Golden Bridge Biotechnology Co, Ltd. (Beijing, China). Lymphocyte Separation Medium was from Haoyang Biological Manufacture Co., Ltd. (Tianjin, China). RPMI-1640 medium, DMEM medium, and FBS were from Gibco (NY, USA). DNP-BSA was from Biosearch (CA, USA). Toluidine blue was from Amresco (Ohio, USA). Antibodies against Syk, p-Syk (Y525/526), PKC, p-PKC, NF-κB p65, p-NF-κB p65 (Ser536), cPLA2, p-cPLA2 (Ser505), and β-actin were obtained from Cell Signaling Technology (MA, USA). The hemocytometer was from Mindray BC-5000 vet (Shenzhen, China). The inverted research microscope was from Nikon Eclipse Ti2 (Japan). PLE was prepared as described in previous study, and the major compounds in PLE were roseoside, vicenin-2, and rosmarinic acid [17 (link)].
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