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7% sodium deoxycholate, and 1% NP-40) and immune complexes were eluted from beads with elution buffer (50?mM Tris-HCl [pH 8.0], 10?mM EDTA, 1% SDS) at 65 C��. Eluates were additionally incubated at 65C�� to reverse crosslinking and then incubated with 0.5?mg/ml Proteinase K at 55 C��. DNA was purified with MinElute PCR purification kit (QIAGEN). The immunoprecipitated DNA regions were quantified by real time PCR using SNS-032 mw ABI Prism. (See also Table S1.) The perigonadal fat pads were fixed in 4% paraformaldehyde in PBS and embedded in paraffin. The sections were incubated with rat F4/80 antibody (Serotec) (1:250 dilution) and goat IL-6 antibody (Santa Cruz) (1:50 dilution) at 4��C, followed by incubation with anti-rat IgG RITC (Santa Cruz) and anti-goat IgG FITC (Santa Cruz) for 1?hr at room temperature. Hoechst click here staining (1:400 dilution) was performed for 20?min at room temperature. The sections were mounted with Fluorescent Mounting Medium (DAKO) and examined under a fluorescence microscope (BZ-8000) (KEYENCE). Blood samples were collected by tail bleed. Plasma adiponectin and IL-6 concentrations were quantified by ELISA assay (Ohtuka Pharmaceuticals and R&D Systems, respectively). Bone marrow cells were collected by flushing of the femurs and tibiae of the mice at 6?weeks of age. The nucleated cells were counted and injected intravenously into lethally irradiated (10 Gy) male IL-6 KO mice at 6?weeks of age. The mice were maintained under normal chow diet for 8?weeks before experiments. For chimerism assay, the genomic DNA purified from the blood samples was subjected to Real-time PCR as previously described (Ichikawa et?al., 2008). Statistical analysis was performed by two-sample t test assuming unequal variances or paired two-sample t test for means. Statistical significance was accepted at p?Enol Priority Areas (S) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to?T.K.), a Grant-in-aid for Scientific Research from the Ministry of Health, Labor, and Welfare (to K.U.), Health Science Research grants (Research on Human Genome and Gene Therapy) from the Ministry of Health and Welfare (to T.K.), and a grant from Takeda Science Foundation (to K.U.). ""Adipocytes are specialized cells that store excess energy in the form of triglycerides and also serve an endocrine function, secreting adipokines that influence systemic energy homeostasis (Halaas et?al., 1995, Steppan et?al., 2001?and?Yamauchi et?al., 2001).