PMC:3279418 / 4735-7230 JSONTXT

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The endogenous expression of Sharpin mRNA in BMDC was determined by quantitative real time-PCR (qRT-PCR) following culture in medium only or after stimulation with LPS. Sharpin mRNA was present in BMDC generated from WT mice (Fig. 1D) and its level was modestly decreased by LPS stimulation. There was a significant reduction of Sharpin mRNA (6–7-fold) in BMDC generated from cpdm mice. Transfection of Flag-tagged Sharpin in fibroblasts (NIH3T3) and macrophages (RAW264.7) indicated cytoplasmic localization of the SHARPIN protein (Fig. 1E).\n10.1371/journal.pone.0031809.g001 Figure 1 In vivo and in vitro features of SHARPIN.\n(A) COILS and MotifScan programs were used to predict the presence of CC (coiled-coil) domain, UBL (ubiquitin-like) domain and ZFRBP (zinc-finger Ran-Binding protein 2) domain which form similar motif patterns in the SHARPIN protein of human, mouse and rat origins. (B) Eight week-old females of WT and cpdm mice. The mutant mice (above) develop progressive skin inflammation starting at about four weeks. (C) Extramedullary hematopoiesis causes marked enlargement of the spleen of cpdm mice. (D) BMDC were incubated in the absence or presence of 100 ng/ml LPS for 4 hours. The mRNA level of Sharpin was measured by qRT-PCR and presented relative to the mRNA expression in non-stimulated WT BMDC. Bars represent the mean ± s.d. of 3 mice. * P\u003c0.05; ** P\u003c0.001. (E) Fibroblast (NIH3T3) and macrophages (RAW264.7) cells were transfected with the expression plasmid pFLAG-SHARPIN. After 48 hours, cells were fixed and probed with anti-FLAG and FITC-conjugated secondary antibody. Nuclei were stained with DAPI. 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