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. 2008 May 12;205(5):1077–1086. doi: 10.1084/jem.20072416

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© 2008 Machado et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 6. — The antiinflammatory actions of ATLs require SOCS2-dependent proteasomal degradation of TRAF2 and TRAF6. (A) WT and SOCS2-deficient mice (n = 10 mice/group) were treated orally with ASA (5 mg/kg), or PBS as a control. 45 min later, splenic DCs were purified and stimulated with LPS (1 μg/ml) or medium alone. At the indicated times, TRAF2, TRAF6, ERK2, p38, JNK2, IκBα, pJNK, pERK1/2, pP38, and actin expression were quantified as indicated in Fig. 2. (B) Splenic DCs purified from WT, SOCS2-deficient, and COX1-deficient mice were incubated in the absence or presence of 5 mM ASA for 0, 30, or 90 min, followed by quantification of actin, TRAF6, and SOCS2 expression by Western blot. (C) Splenic DCs, purified from WT and SOCS2-deficient mice that had been treated with ASA or PBS as in A, were stimulated with LPS (1 μg/ml) or PBS. After overnight culture, IL-12p70 and TNF secretion was quantified in by ELISA. (D) Splenic DCs purified from WT and SOCS2-deficient mice that had been treated with ASA or PBS as in A underwent Western blot analysis of polyubiquitylated TRAF2 and TRAF6, as indicated in Fig. 2. (E and F) WT and SOCS2-deficient mice were injected intraperitoneally with 0.2 mg/Kg PBS or PR11, followed by PBS or ASA treatment. 1 h later, splenic DCs were purified and whole-cell extracts underwent immunoblot analysis to quantify TRAF2 and TRAF6 expression (E). WT and SOCS2-deficient mice were injected intraperitoneally with PR11 (or PBS), followed by ASA (or PBS). 45 min later, mice were challenged intraperitoneally with LPS (5 μg/mouse). 3 h later, serum TNF concentrations were quantified by ELISA (F). (G–O) WT (G, I, K, and M) and SOCS2-deficient (H, J, L, and N) DCs, purified from PBS-treated (F–I) or ASA-treated (J–M) mice, were incubated for 45 min with PBS (G, H, K, and L) or LPS (1 μg/ml; I, J, M, and N), and then cytospun onto slides. DCs subsequently underwent immunofluorescence staining for TRAF6 (green) and p65 (red), followed by counterstaining with DAPI (blue). Microphotographs were taken using an Axiovert microscope with AxioVision software (see Materials and methods). (O) Nuclear extracts were prepared and the levels of DNA-bound p65 quantified using an NF-κB p65 Transcription Factor Assay kit. Data shown are the mean ± the SD of triplicate samples, and are representative of three independent experiments with similar results. *, P < 0.01, PR11 or ASA versus PBS treatment. Bars, 2 μm.

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