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. 2008 Jun 9;205(6):1491–1503. doi: 10.1084/jem.20071728

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© 2008 Cao 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.jgp.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. — MKP-1 mediates the effects of acetylation upon phosphorylation of p38 and NOS2 expression in cells. (A) MKP-1 siRNA decreases MKP-1 expression in RAW cell lines. RAW cells were stably transfected with a vector encoding an siRNA hairpin sequences directed against MKP-1 nucleotides 67–85 (clone #1) or MKP-1 nucleotides 743–761 (clones #2-3) or a control siRNA sequence. Three separate stably transfected clones were isolated, and cell lysates were immunoblotted with antibody to MKP-1. (B) Knockdown of MKP-1 restores phospho-p38 levels and NOS2 expression in RAW cells treated with TSA. RAW cells stably transfected with siRNA directed against MKP-1 were treated with LPS and TSA, and cell lysates were immunoblotted with antibodies to NOS2 or MAPK family members. (C) Cells from MKP-1^−/− mice maintain levels of phospho-p38 after treatment with TSA. MEFs from MKP-1^−/+ or MKP-1^−/− mice were treated with LPS and TSA for 30 min, and cell lysates were immunoblotted with antibodies to p38 as above. (D) Effects of TSA on p38 are restored by rescue of MKP-1^−/− cells with MKP-1(WT), but not with MKP-1(K57R). Cells from MKP-1^−/− were immortalized with SV40 T-antigen, and then transfected with plasmids expressing MKP-1(WT)-ERα or MKP-1(K57R)-ERα. The cells were treated with 4-HT to induce MKP-1-ERα expression, and then treated with LPS and TSA for 1 h. Cell lysates were immunoblotted with antibody to phospho-p38 (top), total p38 (middle), and the ER tag of MKP-1 (bottom). (E) Antiinflammatory effects of TSA are restored by rescue of MKP-1^−/− cells with MKP-1(WT), but not with MKP-1(K57R). Cells from MKP-1^−/− were transfected with plasmids expressing MKP-1(WT)-ERα or MKP-1(K57R)-ERα, treated with 4-HT, and then treated with LPS and TSA for 1 h. Total cell RNA was analyzed by RT-PCR for IL-6, TNF-α, and GAPDH. (F) MKP-1 mediates TSA inhibition of p38 in primary macrophages. Peritoneal macrophages were isolated from WT and MKP-1^−/− mice, stimulated with LPS, TSA, or both, as above, and cell lysates were immunoblotted with antibody to total or phosphorylated p38.