membrane (Invitrogen). The membranes were incubated with primary antibodies overnight at 4uC, followed by incubation with secondary antibodies conjugated with HRP for 1 hour at room temperature. The antibody-bound proteins were detected using a Super Signal West Dura substrate (Pierce) with an OptiChemHR Imaging System (UVP). In vitro kinase assay J774 cells or BMDM were lysed in 600 ml of cell lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1% Triton X-100, 2.5 mM sodium pyrophosphate, protease inhibitor cock- tail). Cell lysates were immunoprecipitated with either JNK, IKKe, or ERK1/2 antibodies overnight at 4uC, and then incubated further with protein A Sepharose for 1 h at 4uC. Immune complexes were washed twice with the lysis buffer and then washed twice with a kinase buffer (20 mM HEPES, pH 7.4, 1 mM MnCl2, 5 mM MgCl2, 10 mM b-glycerophosphate, 0.1 mM sodium orthovanadate, 2 mM NaF, 1 mM DTT). Samples were incubated with 5 mCi of [c-32P]-ATP (NEN Life Science) and 2 mg of purified GST-c-Jun (1–79 aa) or GST-c-Fos (300–380 aa) in 25 ml of kinase buffer for 30 min at 30uC. (The GST-c-Jun and c-Fos cDNA were kindly provided by Michael Karin, UC San Diego). The reactions were stopped by adding LDS sample buffer (Invitrogen), the samples were run on a Nu- PAGE gel (Invitrogen), and phosphorylated proteins were detected by autoradiography. Oil red O staining BMDM were stained for neutral lipid with Oil Red O and hematoxylin, as described previously [5]. Briefly, cells were incubated with media alone or 50 mg/ml mmLDL plus 200 mg/ ml native LDL. After 40 hours, cells were washed, fixed and stained with Oil Red O and hematoxylin. Statistical analysis Graphs represent means 6 standard error from 2–4 indepen- dent experiments. Significance of differences was calculated using one-way ANOVA. Results Generation of myeloid cell-specific Syk knockout mice Syk total knockout mice are perinatally lethal [1]. Thus, we have generated myeloid cell-specific Syk knockout mice by breeding Sykflox/flox mice with LysM-Cre mice. mRNA expression of Syk was undetectable by RT-PCR in BMDM from Syk2/2 mice (Sykflox/flox Cre+) compared to WT littermates (Sykflox/flox Cre2) (Fig. 1A). Syk protein levels in both peritoneal macrophages and BMDM were reduced by 60–70% (Fig. 1B; see also Figs. 2B and S1). Because in our earlier studies with Syk shRNA J774 cells we demonstrated that Syk knockdown decreased macrophage lipid accumulation [5], we tested lipid accumulation in Syk2/2 BMDM. As expected, lipid accumulation in Syk2/2 BMDM was dramatically reduced compared to WT BMDM (Fig. 1C). These results confirm that primary Syk2/2 macrophages have a phenotype similar to that observed in a Syk knockdown cell line. Syk regulates mmLDL-induced activation of AP-1 An initial step in TLR4 activation is ligand-dependent homodimerization of TLR4. Thus, we tested whether mmLDL induced dimerization of TLR4, using the established model of Ba/ F3 cells stably expressing GFP-TLR4 and Flag-TLR4 [13,14]. As shown in Fig. 2A, in cells incubated with mmLDL, GFP-TLR4 Syk Regulates AP-1 Dependent Response to mmLDL Figure 1. Reduced Syk expression in macrophages from Sykflox/ flox/LysM-Cre mice. (A) Total RNA was isolated from BMDM of Sykflox/ flox/LysM-Cre(2) (WT) and Sykflox/flox/LysM-Cre(+) (Syk2/2) mice, and RT- PCR with Syk and GAPDH primers was performed. (B) Lysates of resident peritoneal macrophages (PM) and BMDM were run on SDS- PAGE, transferred to a membrane and immunoblotted with anti-Syk and anti-GAPDH antibodies. (C) WT and Syk2/2 BMDM were incubated with 50 mg/ml mmLDL (to induce macropinocytosis) and 200 mg/ml of native LDL (lipid carrier) for 40 hours. The cells were stained for neutral lipid with Oil Red O. doi:10.1371/journal.pone.0032378.g001 pulled down Flag-TLR4, indicating mmLDL-induced TLR4 dimerization. In addition, TLR4 pulled down Syk as well, reiterating our earlier findings of mmLDL-induced Syk recruit- ment to TLR4 [4,5]. Blocking TLR4/MD-2 with a specific antibody diminished mmLDL-induced recruitment of Syk to TLR4 (Fig. 2A). Next, we used primary Syk-deficient macrophages to evaluate Syk-dependent pathways downstream from TLR4. In BMDM, mmLDL induced phosphorylation of ERK1/2, JNK and c-Jun, and the Syk deficiency dramatically reduced mmLDL-induced phosphorylation of these signaling proteins (Fig. 2B). We also tested Sykflox/flox peritoneal macrophages infected with a Cre adenovirus and confirmed that mmLDL-induced ERK1/2 phosphorylation was Syk-dependent (Fig. S1). mmLDL did not induce phosphorylation of p65 in macrophages (Fig. S2), validating earlier findings that mmLDL does not activate the NF-kB pathway [19]. Phosphorylated c-Jun and c-Fos are major components in the assembly of a heterodimeric AP-1 transcriptional complex. c-Jun is phosphorylated by JNK and IKKe [20–22]. Indeed, using an in vitro kinase assay, we demonstrated that both JNK and IKKe phosphorylated c-Jun (Figs. 2C and S3). Both phosphorylation events were absolutely dependent on Syk (Fig. 2D). c-Fos is phosphorylated by activated ERK1/2 and p38 [23,24]. Our previous studies have shown that mmLDL activates ERK1/2, but not p38 [5,17]. Thus, we performed an in vitro kinase assay in lysates precipitated with an ERK1/2 antibody and found that in mmLDL-activated macrophages, ERK1/2 phosphorylated c-Fos and that this process was Syk-dependent as well (Fig. 2C,D). Next, we tested c-Jun and c-Fos binding to consensus DNA sites using a PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e32378