Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin, and 0.25 % (
w/
v) trypsin/EDTA were obtained from GIBCO (Gaithersburg, MD). Endotoxin-free fetal bovine serum was from Atlanta Biologicals (Lawrenceville, GA). Lipopolysaccharide (LPS) (rough strains) from
Escherichia coli F583 (Rd mutant) was purchased from Sigma-Aldrich (St. Louis, MO). Interferon-γ (IFNγ) was purchased from R&D Systems (Minneapolis, MN). Pharmacological inhibitors used include the following: U0126, SB202190, and SP600125 were from Cell Signaling (Beverly, MA). Arachidonyl trifluoromethyl ketone (AACOCF3), pyrrophenone, racemic bromoenol lactone (BEL), nordihydroguaiaretic acid (NDGA), ibuprofen, zileuton, and PD146176 were from Cayman Chemical (Ann Arbor, MI). NCTT-956 was from Sigma-Aldrich (St. Louis, MO). RNA interference Lipofectamine RNAiMAX Transfection Reagent was from Life Technology (Carlsbad, CA). siRNA against cPLA
2 Mm_Pla2g4a_8 FlexiTube siRNA (NM_008869) and AllStars Negative Control siRNA were purchased from Qiagen (Hilden, Germany). Antibodies used for Western blots include the following: goat anti-rabbit IgG-horseradish peroxidase, goat anti-mouse IgG-horseradish peroxidase, anti-cPLA
2 rabbit polyclonal, anti-iNOS rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal anti-β-actin peroxidase (Sigma-Aldrich, St. Louis, MO); rabbit polyclonal anti-p-cPLA
2, rabbit polyclonal anti-ERK1/2, and mouse monoclonal anti-phospho-ERK1/2 antibodies (Cell Signaling, Beverly, MA). An affinity-purified antibody directed against an iPLA
2β peptide corresponding to residues 277–295 was a gift of Drs. Chris Jenkins and Richard Gross (Washington University School of Medicine, St. Louis, MO) [
33]. For immunocytochemical staining, rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1) antibodies (019–19741) was purchased from Wako BioProducts (Richmond, VA), Alexa Fluor 488® phalloidin from Life Technologies (Carlsbad, CA), and 4′,6-diamidino-2-phenylindole (DAPI) from Roche Molecular Chemicals (Basel, Switzerland). For ROS detection, CM-H2DCF-DA (DCF) was purchased from Invitrogen, Inc. (Eugene, OR). WST-1 assay was purchased from Clontech (Mountain View, CA). Prostaglandin E2 (PGE2) EIA Kit was purchased from Cayman Chemicals (Ann Arbor, MI).