object on a Nikon SCLIPSS TE2000-S microscope (Nikon) equipped with ACT-1 software (Nikon). Original magnification was 4006. Western blot analysis Six-week-old female BALB/c mice were injected intramuscu- larly with pSAP (100 mg/mice). 10 days later, the serum was collected and western blot analysis was performed as described previously [37]. In briefly, serum was electrophoresed on SDS- PAGE gels and then transferred to the PVDF membrane. The membrane was probed with rabbit anti-SAP (Calbiochem), mouse anti-FLAG (Santa Cruz), or rabbit anti-GAPDH antibody (Santa Cruz), followed by HRP-conjugated goat anti-rabbit antibody (Santa Cruz) or goat anti-mouse antibody (Southern Biotech). The signals were developed by chemiluminescence (Pierce). Binding ability of SAP to DNA The binding ability of SAP to DNA was detected by dot blot analysis with mouse SAP protein purified from pSAP treated mice and rabbit anti-mouse SAP (Calbiochem) as previously described [38]. In briefly, DNA (1 mg) was spotted on the nitrocellulose membranes. After the incubation of SAP protein (1 mg/ml), anti- SAP Abs, and peroxidase-labeled IgG Abs (Southern Biotech), the blots were developed with 3, 39-Diaminobenzidine (DAB) to measure the binding ability of SAP to DNA. Quantitative analysis of blots was done using Mini-Transilluminator (Bio-Rad) equipped with molecular analysis software. The binding ability of SAP to DNA was reflected as mean intensity. DNA uptake in vitro ALD-DNA was labeled with Alexa Fluor 488 (Invitrogen) according to the manufacturer’s instructions. The labeled ALD- DNA (referred as AF488-ALD-DNA) was purified using Bio-Rad Micro Bio-Spin P-30 column (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. AF488-ALD-DNA was incubated with purified mouse SAP protein (SAP plus ALD-DNA) at 37uC for 2 h. BMDMs were treated with chloroquine (100 mg/ml) before DNA incubation. The intracellular Alexa Fluor 488 labeled ALD-DNA (AF488-ALD-DNA) was determined by flow cytometry (FACSCa- libur) as previously described [39]. All flow cytometry data were acquired on a BD FACSCalibur (BD Biosciences) in CellQuest (BD Biosciences) and analyzed by FlowJo software (Tree Star). Real-time PCR analysis Total RNA was isolated from cultured cells with TRIzol reagent (Invitrogen) and was reverse-transcribed (RT) using a cDNA synthesis kit (MBI Ferments) according to the manufacturer’s instructions. Subsequently, cDNA was subjected to quantitative real-time PCR using a Lightcycler480 and SYBR Green system (Roche Diagnostics) following the manufacturer’s protocol [40]. References 1. Rahman A, Isenberg DA (2008) Systemic lupus erythematosus. N Engl J Med 358: 929–939. 2. Walport MJ (2000) Lupus, DNase and defective disposal of cellular debris. Nat Genet 25: 135–136. 3. Kotzin BL (1996) Systemic lupus erythematosus. Cell 85: 303–306. 4. Savill J, Dransfield I, Gregory C, Haslett C (2002) A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2: 965–975. 5. Hoffmann MH, Trembleau S, Muller S, Steiner G (2010) Nucleic acid-associated autoantigens: pathogenic involvement and therapeutic potential. J Autoimmun 34: J178–206. 6. Qiao B, Wu J, Chu YW, Wang Y, Wang DP, et al. (2005) Induction of systemic lupus erythematosus-like syndrome in syngeneic mice by immunization with activated lymphocyte-derived DNA. Rheumatology (Oxford) 44: 1108–1114. Reversal of Lupus Nephritis by SAP Treatment Flow cytometry analysis Murine renal tissues were surgical resected and dispersed in RPMI 1640 contained 5% FBS and 0.1% collagenase (Sigma- Aldrich) at 37uC for 30 min, followed by progressive sieving to obtain single-cell suspensions. To assess the infiltration of leucocyte populations in kidneys of mice, flow cytometry analysis were performed with PE-labeled anti-CD45, PerCP-labeled anti-CD4, FITC-labeled anti-CD19, and FITC-labeled anti-F4/80 (BD Biosciences). All flow cytometry data were acquired on a BD FACSCalibur (BD Biosciences) in CellQuest (BD Biosciences) and analyzed by FlowJo software (Tree Star). ELISA Assay To assess protein levels of TNF-a, IL-1b, IL-6, IL-10, IL-12, and MCP-1 in the homogenized kidney tissue and in serum of mice, ELISA assays were performed with relative ELISA Kits (eBioscience) according to the manufacturer’s instructions. Pathological analysis For histology analysis, murine renal tissues were surgical resected and fixed in 4% paraformaldehyde (Sigma-Aldrich), processed, and embedded in paraffin. H&E staining of renal tissue sections were performed according to the manufacturer’s instructions and assessed by a pathologist blinded to treatment group. The kidney score of glomerulonephritis was determined by using the ISN/ RPS2003 classification. Fluorescent staining of cryosections was used for autoantibody deposition analysis in the glomeruli. Sections were fixed in acetone for 10 min and incubated with FITC- conjugated goat anti-mouse IgG (H+L chain specific) Ab (Sigma- Aldrich) for 30 min. Pictures were acquired with Nikon SCLIPSS TE2000-S microscope (Nikon) equipped with ACT-1 software (Nikon). Original magnification was 6200. Statistical analysis All data are expressed as means 6 SD of three independent experiments or from a representative experiment of three independent experiments. The statistical significance of the differences in the experimental data was valued by the Student’s t-test. The statistical significance level was set as * P,0.05, ** P,0.01, *** P,0.001. Author Contributions Conceived and designed the experiments: WZ JW SX. Performed the experiments: WZ JW. Analyzed the data: WZ JW WX. Contributed reagents/materials/analysis tools: WZ JW BQ WX. Wrote the paper: WZ JW BQ SX. 7. Wen ZK, Xu W, Xu L, Cao QH, Wang Y, et al. (2007) DNA hypomethylation is crucial for apoptotic DNA to induce systemic lupus erythematosus-like autoimmune disease in SLE-non-susceptible mice. Rheumatology (Oxford) 46: 1796–1803. 8. Zhang W, Xu W, Xiong S (2010) Blockade of Notch1 signaling alleviates murine lupus via blunting macrophage activation and M2b polarization. J Immunol 184: 6465–6478. 9. Garlanda C, Bottazzi B, Bastone A, Mantovani A (2005) Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annu Rev Immunol 23: 337–366. 10. Bottazzi B, Doni A, Garlanda C, Mantovani A (2010) An integrated view of humoral innate immunity: pentraxins as a paradigm. Annu Rev Immunol 28: 157–183. PLoS ONE | www.plosone.org 12 July 2011 | Volume 6 | Issue 7 | e22659