exhaustive washing with 1 x PBS and the adherent cells were collected for experimental purpose. Western Blot Analysis Total proteins were prepared from BMDCs or splenocytes using RIPA lysis buffer supplemented with protease inhibitors. Western blot analysis was carried out as reported by probing the blots with an indicated primary Ab (Santa Cruz, CA) followed by an HRP- conjugated secondary antibody. The reactive bands were visualized using an ECL PlusTM Western blot kit (PIERCE, Rockford, IL) [55]. b-actin was used for normalization. All Western blotting antibodies were purchased from Santa Cruz. Flow Cytometry Analysis BMDCs or splenic single cell suspensions were prepared and washed with PBS. After blocking with anti-mouse CD16/32 Fcc III/II receptor, the cells were stained for CD11c, CD11b, I-Ad, CD80, CD86, and CD54 as reported [56]. After washes, the cells were suspended in FACS buffer and analyzed on a FACSCalibur (BD Bioscience, San Jose, CA). The data were analyzed using CellQuest v3.3 software as instructed. All flow cytometry antibodies were purchased from BD Bioscience. ELISA Analysis of Cytokines The amount of TNFa, IL-2, IL-10, IL-12, IL-17 and IFN-c in the culture media and sera was determined using the sandwich ELISA kits (eBioscience, San Diego, CA) as reported [57]. Mixed Lymphocyte Reaction (MLR) Splenic DCs originated from Jak22/2 and control mice were purified using a mouse DC enrichment kit (StemCell, Seattle, WA). For allogenic MLR, T cells isolated from BALB/cJ mice were co-cultured with irradiated (2000 rad) BMDCs or splenic DCs (26105/ml, DC:T = 1:10) originated from Jak22/2 or control mice for 56 h followed by additional 16 h in the presence of 0.5 mCi/well 3H thymidine. For antigen-specific MLR, 2.56105/ml irradiated (2000 rad) Jak22/2 BMDCs or splenic DCs were pulsed with 1 mM OVA peptide and then co-cultured with 56105/ml splenocytes isolated from OT-1 transgenic mice as above. After washes, the cells were finally harvested on glass wool filters with a suction-water-wash apparatus, dried, and counted in a beta scintillation counter [57]. T cell proliferation was estimated as mean CPM (counts per minute) 6 SE. Listeria monocytogenes Infection Jak22/2 and control mice were i.v. injected with 16106 CFU (,1 LD50) Listeria Monocytogenes (LM-ova, a modified Listeria strain expressing OVA) in PBS as reported [58,59]. Determination of Listeria Monocytogenes titer was carried out as previously described with minor modifications [59]. Briefly, spleens and livers were harvested after 48 h infection and homogenized in sterile distilled water containing 0.2% triton X-100. After 30 min incubation at room temperature, serial 10-fold dilutions were plated on TSB- agar and incubated at 37uC. Colonies were counted after 48 h incubation. Stat3 siRNA Transfection in DC2.4 Cells DC2.4 cells were transfected with a Stat3 siRNA or a scramble RNA (Santa Cruz, CA) as instructed by the manufacturer. The cells were stimulated with 0.5 mg/ml LPS after 24 h transfection. Cell lysates and culture supernatants were then subjected to Western blot and ELISA analysis after 24 h stimulation, respectively. Jak2 Modulates Innate Immunity Induction of LPS-Challenged Septic Shock For induction of septic shock, the mice were first i.p. injected with 50 mg/kg body weight of LPS (Sigma, St Louis, MO), and then subjected to evaluation of mortality and behavior changes every 6 h for .96 h. Mice injected with 150 mg LPS were used as a model for nonlethal sepsis. Serum samples were collected using whole blood after 12 h of injection. For adoptive transfer studies, both Jak22/2 and control mice were first underwent 800 rads of irradiation. Two days later, 16107 BMDCs derived from nonirradiated control mice were injected into these irradiated mice via tail vein, respectively. After 4 h of transfer, the mice were underwent induction of septic shock with lethal dose of LPS (25 mg/kg body weight) as above. Eight mice were included in each study group. Statistical Analysis Survival curves for septic shock were generated by the Kaplan and Meier method. Chi-square test was employed to determine the difference of mortality for LPS-induced septic shock. Comparisons between groups for flow cytometry, cytokine and MLR data were accomplished by one-way ANOVA using SPS 11.5 for windows. Data were present as mean 6 SE. P,0.05 was considered statistically significant. Supporting Information Figure S1 Loss of Jak2 suppresses the maturation of splenic DCs. Surface marker expressions were analyzed by flow cytometry in splenic DCs. Each bar represents the average percentage of DCs positive for the surface marker analyzed. Found at: doi:10.1371/journal.pone.0009593.s001 (0.12 MB TIF) Figure S2 AG490 suppresses BMDC maturation in a dose- dependent manner. Bone marrow cells derived from B6 mice were used to generate BMDCs. AG490 (0, 100 and 200 mM, respectively) was added into the cultures on day-4. BMDCs were stimulated with LPS (500 ng/ml) on day-9 and harvested on day- 10 for flow cytometry analysis of surface marker expressions. Left panel: a representative flow cytometry data of three independent experiments performed; right panel: a bar graphic figure showing the average percentage of BMDCs positive for each surface marker analyzed. *, p,0.05. Found at: doi:10.1371/journal.pone.0009593.s002 (4.21 MB TIF) Figure S3 Tamoxifen does not have perceptible effect on DC phenotype. 25 mg/kg body weight of tamoxifen or carrier solution were i.p. injected into B6 mice for 5 consecutive days. The mice were sacrificed after 2 wk of last injection. Bone marrow cells were used to generate BMDCs and surface marker expressions were analyzed before/after LPS stimulation. Found at: doi:10.1371/journal.pone.0009593.s003 (0.67 MB TIF) Figure S4 Loss of Jak2 suppresses surface marker expressions in macrophages. Macrophages deficient for Jak2 show less matured phenotype. Each bar represents the average percentage of macrophages positive for each surface marker analyzed. Found at: doi:10.1371/journal.pone.0009593.s004 (0.13 MB TIF) Figure S5 IL-10 secretion after LPS challenge (A) ELISA analysis of IL-10 production of culture supernatants derived from Jak22/2 and wild-type BMDCs before/after LPS (1 mg/ml) treatment. (B) Serum IL-10 levels after LPS challenge. Both Jak22/2 and control mice were first challenged with nonlethal dose of LPS (150 mg/mouse) and 12 h later the mice were sacrificed. Sera were pooled from three mice and then subjected to ELISA analysis of IL-10. PLoS ONE | www.plosone.org 10 March 2010 | Volume 5 | Issue 3 | e9593