purchased from the Jackson Laboratory. Myeloid cell lineage specific Syk knockdown mice, thereafter referred to as Syk2/2, were generated by breeding Sykflox/flox mice with LysM-Cre mice, the latter express myeloid cells specific lysozyme 2 promoter- driven Cre recombinase. Littermate Sykflox/flox/Cre(2) mice, thereafter referred to as wild type (WT), were used as a control in all experiments. In addition, we used MyD882/2 mice on the C57BL6 background [10], kindly provided by Dr. Shizuo Akira from Research Institute for Microbial Diseases, Osaka University, and wild type C57BL6 mice as a control. Cell culture The majority of experiments were performed with bone marrow derived macrophages (BMDM) obtained from bone marrow cells isolated from Sykflox/flox/Cre(+) (Syk2/2) and Sykflox/flox/Cre(2) (WT) mice and differentiated with macrophage colony stimulating factor (L929 conditioned medium) according to published protocols [11]. Briefly, after mice were sacrificed, tibias and femurs were dissected and washed with a BMDM growth media (10% FBS, 50 mg/ml gentamicin and supernatant from L929 cells) to suspend cells. The cells were placed in 10-cm Petri dishes and maintained in a humidified 37uC incubator for 5 days to allow differentiation. Resident peritoneal mouse macrophages were isolated by peritoneal lavage, without any prior elicitation. Isolated peritoneal macrophages were plated with DMEM containing 20% FBS and 50 mg/ml gentamicin. After 3 hours, floating cells were removed and attached cells were further incubated with 0.5% FBS/DMEM overnight. Other cell types in which we achieved Syk loss- or gain-of- function were used to validate the results obtained with Syk2/2 BMDM. Chinese hamster ovary-K1 cells (CHO) were from American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplement- ed with 10% fetal bovine serum (FBS) and 50 mg/ml gentamicin. Murine macrophage-like J774A.1 cell lines stably expressing Syk- specific or scrambled shRNA [5] were cultured in DMEM supplemented with 10% FBS, 50 mg/ml gentamicin and 0.8 mg/ ml of G418 (Calbiochem) to maintain selection. A murine fibroblast cell line stably expressing human 15-lipoxygenase [12] was cultured in 10% FBS and 50 mg/ml gentamicin with 0.5 mg/ ml of G418. Ba/F3 cells stably expressing GFP-TLR4 and Flag- TLR4 [13,14] were cultured in RPMI1640 medium (Invitrogen) containing 70 units/ml recombinant murine interleukin-3, 10% heat-inactivated FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). Antibodies Two monoclonal antibodies to mouse TLR4/MD-2 antibodies, UT12 and UT18, were from eBioscience. An isotype mouse IgG3k control was from BioLegend Antibodies specific to Syk, JNK, and IKKe were from Santa Cruz Biotechnology; ERK1/2, p-ERK1/2, p-JNK, p-c-Jun, p-p65, and GAPDH from Cell Signaling Technology; GFP from Abcam; and Flag from Sigma- Aldrich. LDL preparation and modification LDL (density = 1.019–1.063 g/ml) was isolated from pooled human plasma by sequential ultracentrifugation [15]. Plasma was obtained from normal volunteers according to a protocol approved by the UCSD Human Research Protection Program (Project #071402). Native and modified LDL preparations were tested for possible endotoxin contamination using a Limulus Amoebocyte Lysate kit (Cambrex, Walkersville, MD). Only preparations with endotoxin ,2.5 pg/ml at a final dilution were Syk Regulates AP-1 Dependent Response to mmLDL used in experiments. To produce mmLDL, we incubated 50 mg/ ml of native LDL in serum-free DMEM for 18 hours with murine fibroblasts overexpressing human 15-lipoxygenase [16,17]. To produce OxLDL, 100 mg/ml of native LDL was incubated with 10 mM CuSO4 for 18 hours at 37uC and then concentrated to 1 mg/ml [18]. The extent of LDL oxidation was assessed by measuring thiobarbituric acid reactive substances (typically, more than 30 nmol/mg protein in OxLDL). Recombinant adenovirus Recombinant adenovirus expressing Cre recombinase was kindly provided by Byungkwan Lim (Kirk Knowlton’s lab, UC San Diego). Recombinant adenoviruses were expanded in HEK293 cells (from ATCC). Isolated peritoneal macrophages were infected with recombinant adenovirus expressing Cre recombinase at 500 multiplicity of infection (MOI) for 48 hours. A recombinant adenovirus expressing GFP was used as control. After infection, cells were treated with media or mmLDL (50 mg/ ml) and then cells were harvested. Real-time quantitative PCR (qPCR) Total RNA was isolated from 16106 cells using RNeasy columns (Qiagen). Isolated RNA was reverse transcribed using a First Strand Synthesis kit (Invitrogen) following manufacturer’s protocol. Probes, primers and reagents for TagMan qPCR were purchased from Applied Biosystems. qPCR analysis was per- formed using a Rotor Gene Q thermocycler (Qiagen). Cytokine ELISA Cells (0.26106) were plated overnight and then incubated with media alone (DMEM supplemented with 5% lipoprotein-deficient serum (LPDS)) or in the presence of 50 mg/ml mmLDL for 6 hours or 24 hours. Supernatants were collected and centrifuged at 10,000 rpm for 5 min to remove floating cells. Levels of MIP-2 (CXCL2) and IL-6 secreted proteins were measured in ELISA using reagents from R&D Systems. Chemiluminescent assay for transcription factor/DNA binding A chemiluminescent transcription factor/DNA binding assay was performed as described previously [19]. Briefly, cellular nuclear extracts were isolated using a Nuclear Extraction kit (Active Motif). The DNA binding of transcription factors (c-Jun, c- Fos, and p65) was measured using an Active Motif’s TransAM kit (Active Motif). Luciferase reporter assay CHO cells were transfected with 1 mg of Syk expression plasmid (kindly provided by Yun Soo Bae, Ewha Womans University, Korea), 0.2 mg of AP-1-luciferase plasmid (provided by Byungk- wan Lim and Kirk Knowlton, UC San Diego), and 0.2 mg of b- galactosidase plasmid. Thirty six hours after transfection, cells were stimulated with 50 mg/ml mmLDL or media alone for 6 hours. Luciferase activity was normalized to b-galactosidase activity. Immunoblot analysis Cells were lysed with an ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM Na3VO4, 1 mM NaF, and protease inhibitor cocktail from Sigma). Protein content was measured by a DC protein assay kit (BioRad) and equal protein amounts were loaded and run on a 4– 12% PAGE gel (Invitrogen) and then transferred to a PVDF PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32378