Journal: Arthritis Research & Therapy

Article Title: Anti-PM/Scl antibodies are found in Japanese patients with various systemic autoimmune conditions besides myositis and scleroderma

doi: 10.1186/s13075-015-0573-x

Figure Lengend Snippet: Detection of anti-PM/Scl antibodies in immuoprecipitation analysis. TnT-IPP: immunoprecipitation of biotinylated recombinant PM/Scl-100 and PM/Scl-75. Recombinant proteins were subjected to 4% to 20% SDS-PAGE and analyzed by immunoblotting with streptavidin-alkaline phosphatase and substrate. In., the input was half the dose for immunoprecipitation. Lanes A to K correspond to the anti-PM-Scl-100 and/or −75-positive patients shown in Figure . Lane N: healthy control serum. HeLa-IPP: immunoprecipitation analysis using radiolabeled HeLa cell extracts. Lanes A to L correspond to the patients shown in Figure and Table . Lanes A to K correspond to anti-PM-Scl-100 and/or −75-positive patients shown in Figure . Lane M: [Methyl- 14 C] methylated protein MW markers (PerkinElmer Japan, Yokohama, Japan). Lane L: anti-U1-RNP-positive serum with equivocal titers for both antibodies in ELISA. Lanes 1 to 4 show the reference sera; lane 1, anti-PM/Scl-positive serum; lane 2, anti-MDA5-positive serum; lane 3, anti-TIF1-γ-positive serum; lane 4, anti-Mi-2-positive serum. Arrow and arrowhead corrspond to the PM/Scl-100 and PM/Scl-75 antigens, respectively. IPP, immunoprecipitation; TnT, in vitro translation and transcription product.

Article Snippet: Lane M: [Methyl- 14 C] methylated protein MW markers (PerkinElmer Japan, Yokohama, Japan).

Techniques: Immunoprecipitation, Recombinant, SDS Page, Western Blot, Control, Methylation, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Scientific reports

Article Title: The transcription factor MafB promotes anti-inflammatory M2 polarization and cholesterol efflux in macrophages.

doi: 10.1038/s41598-017-07381-8

Figure Lengend Snippet: Figure 3. MafB facilitates cholesterol efflux by upregulating ATP-binding cassette transporters. (a,b) Effect of ectopic expression (a) or siRNA-mediated knockdown (b) of MafB on expression of Abca1 and Abcg1 in mouse BMDM (RT-PCR assay). Expression of ABC transporters was further induced using RXR and LXR agonists, 9cRA (0.5 µM) and GW3965 (1 μM), respectively. Expression levels are presented relative to expression by mock- and/or vehicle-treated control. *p < 0.05 (n = 4). (c) Effect of ectopic expression of an indicated gene on ABC transporter expression in RAW 264.7 cells (RT-PCR). Data are presented as in a. All: p < 0.05 (n = 4). (d) Effect of indicated gene overexpression on cholesterol efflux in J774A.1 cells. All: p < 0.05 (n = 6). (e–g) Effects of MafB, MafB (ΔBD) and c-Maf on activation of ABCA1 and FASN promoters in 293ETN cells. Luciferase activity of cells transfected with a reporter of human ABCA1 (e), LXRE (f) or FASN (g), with or without priming with LXRα co-expression. At 24 h after transfection, a LXRα agonist T0901317 (T09) (1 µM) was treated for

Article Snippet: Flag-tagged MafB was purified with M2 Affinity Gel as previously described5, and Myc/DDK-tagged LXRα was obtained from OriGene.

Techniques: Binding Assay, Expressing, Knockdown, Reverse Transcription Polymerase Chain Reaction, Control, Over Expression, Activation Assay, Luciferase, Activity Assay, Transfection

Journal: Scientific reports

Article Title: The transcription factor MafB promotes anti-inflammatory M2 polarization and cholesterol efflux in macrophages.

doi: 10.1038/s41598-017-07381-8

Figure Lengend Snippet: Figure 5. Global view of regulation of MafB expression and its therapeutic implications. (a,b) Integrative analyses of gene expression (RT-PCR, n = 4). Agonist used for each receptor is as follows. LXR: GW3965, RXR: 9cRA, TLR3: poly(I:C), TLR4: LPS, TLR7: imiquimod, and TLR9: CpG-ODN. Unless otherwise specified, results obtained in mouse BMDM were shown. Dex(h) indicates dexamethasone-treated human MDM, in which MRC1 expression was measured instead of Arg1. (a) Heatmap analysis. Row: different stimuli, Column: different genes. Log2 transformation of median fold changes (stimulation versus vehicle-treated) of expression levels of indicated genes were plotted. (b) Principal component analysis (PCA) and stimulation contribution plot. Principal component one (PC1) and two (PC2) primarily capture cholesterol efflux and M1/M2 polarization, respectively. Black: each gene in the PC space. Blue: contribution of each stimulus to the PCs. (c) Effect of MafB deficiency on c-Maf expression (RT-PCR). Mafb+/− and Mafb−/− MEFs were compared (*p < 0.05). Increase IRF7 was shown as a positive control.

Article Snippet: Flag-tagged MafB was purified with M2 Affinity Gel as previously described5, and Myc/DDK-tagged LXRα was obtained from OriGene.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Positive Control