Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Biochemical Isolation of the Myddosome from Murine Macrophages

doi: 10.1007/978-1-4939-7519-8_6

Figure Lengend Snippet: Myddosome formation induced by LPS in wild-type (WT) iBMDMs. Cells were stimulated with 1 μg/mL LPS for indicated time points (30, 60, and 120 min), and were subsequently lysed. Myddosomes were isolated from cleared lysates by MyD88 immunoprecipitation. Components of the myddosome eluates (IRAK2, IRAK4, TIRAP, and MyD88) and input samples were separated by SDS- PAGE and were detected by western blotting. The membrane was developed with the Biorad ChemiDoc™ XRS+ System. Note: Asterisk (*) indicates the IgG light chain [IgG(L)]

Article Snippet: Antibodies and Imaging Tools Primary antibodies for blotting myddosome components: IRAK2 (anti-IRAK2, Rabbit polyclonal, Prosci, 3595).

Techniques: Isolation, Immunoprecipitation, SDS Page, Western Blot, Membrane

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: Primers used for qRT-PCR analysis.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques:

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: ( A ) Representative Mac-3 staining of aortic sinus plaques of placebo-, fenofibrate- and rosiglitazone-treated DKO mice at 24 weeks. ( B ) Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Tnfα , IL6 , Mcp1 and Irak3 . Data are means ± SEM. Scale bar = 500 µm. * P <0.05, ** P <0.01 and *** P <0.001 DKO compared with C57BL/6 J mice; $$ P <0.01 and $$$ P <0.001 PPAR agonist-treated compared with placebo-treated DKO mice; £ P <0.05, ££ P <0.01 and £££ P <0.001 rosiglitazone-treated compared with fenofibrate-treated DKO mice.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques: Staining, Gene Expression, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: ( A ) Soluble Mcp1 protein levels in DKO BMDM exposed to 50 µM fenofibrate, 10 µM rosiglitazone or 5 µM GW9662 for 24 hours as determined by ELISA. Data are means ± SEM; n = 16 from three different mice. $$ P <0.01 compared with fenofibrate-treated BMDM. ( B ) Irak3 RNA and protein levels of DKO BMDM exposed to 1 or 10 µg/mL globular adiponectin for 24 hours as determined by qRT-PCR and Western blotting. Data are means ± SEM; n = 6. *** P <0.001 compared with DKO BMDM; $ P <0.05 and $$ P <0.01 compared with DKO BMDM exposed to 1 µg/mL globular adiponectin. ( C ) Soluble Mcp1 protein levels (n = 18 from three different mice), NFκB p50 DNA binding activity (n = 8 from two different mice) and mROS production (n = 6) in IRAK3 −/− BMDM exposed to 50 µM fenofibrate or 10 µM rosiglitazone for 24 hours as determined by ELISA and flow cytometry. Data are means ± SEM. * P <0.05, ** P <0.01 and *** P <0.001 compared with C57BL/6 J BMDM; $ P <0.05 and $$$ P <0.001 compared with IRAK3 −/− BMDM; £££ P <0.001 compared with fenofibrate-treated BMDM. Abbreviations: BMDM, bone marrow-derived macrophages; mROS, mitochondrial reactive oxygen species.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Binding Assay, Activity Assay, Flow Cytometry, Derivative Assay

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: Plaque volume was determined by measuring lipid (oil red O)-stained surfaces in subsequent sections; macrophages were stained with anti-Mac-3 antibody. Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Pparγ , Mcp1, Irak3 and Adipoq . Data are means ± SEM. ** P <0.01 and *** P <0.001 HFD-fed compared with SD-fed LDL-receptor deficient mice. Abbreviations: HFD, high fat diet; SD, standard diet.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques: Staining, Gene Expression, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: The schematic draw demonstrates the anti-atherosclerotic properties of the PPARγ agonist rosiglitazone. Treatment with rosiglitazone improves the adipocyte function characterized by a decrease in adipocyte size, a reduction in adipose tissue macrophages and an increased expression of anti-inflammatory adiponectin. The increase in blood adiponectin and de novo adiponectin production in atherosclerotic lesions is necessary for the upregulation of Irak3 in plaque macrophages, which is crucial for the indirect rosiglitazone-mediated decrease in Mcp1 secretion. Abbreviations: Mφ, macrophages; ROS, reactive oxygen species.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques: Expressing