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. Author manuscript; available in PMC: 2016 Oct 19.
Published in final edited form as: J Immunol. 2011 Oct 31;187(11):6108–6119. doi: 10.4049/jimmunol.1100373

FIGURE 7. Absence of Sdc1 rescues the anti-inflammatory phenotype of Dcn−/− mice.

FIGURE 7

(A,B) Genotyping of Dcn−/−/Sdc1−/− knockout mice. (A) Genomic PCR showed no Dcn signal (162 bp) for the Dcn−/− and the Dcn−/−/Sdc1−/−, but the respective knock-out signal of 250 bp. (B) Genomic PCR showed no Sdc1 signal (250 bp) for the Sdc1−/− and the Dcn−/−/Sdc1−/−, but the respective knock-out signal of 450 bp. (C) Western blotting reveals expression of DCN in extracts or wild-type (WT) and Sdc−/− mouse ear tissue, and absence in Dcn−/− and Dcn−/−/Sdc1−/− mouse ear tissue. (D) Immunohistochemistry demonstrates presence of SDC1 expression in the epithelial layer of wild-type mouse ears, and absence of SDC1 expression in Dcn−/−/Sdc1−/−mouse ears. 20x magnification. (E) Immunofluorescence microscopy demonstrates presence of DCN expression in the interstitial matrix of wild-type mouse ears, and absence of DCN expression in Dcn−/−/Sdc1−/− mouse ears. 20 x magnification. (F) Dcn−/−/Sdc1−/−, wild-type and Dcn−/− mice were employed in a DTH assay as described in Figure 1 to evaluate the contribution of Sdc1 to the phenotype of Dcn−/− mice. Dcn−/− mice show a reduced response to DTH compared to wild-type (cf. Fig. 1). Dcn−/−/Sdc1−/−mice respond to oxazolone like the wild-type mice. Compared to the Dcn−/− mice, the response is significantly different, indicating that Dcn-dependent modulation of Sdc1 contributes to the DTH response in vivo (n=5; ** P < 0.01). (G) Quantitative real-time PCR analysis for ICAM-1 expression in ear tissue of wild-type, Dcn−/− and Dcn−/−/Sdc1−/−mice 24h +/− DTH eliciation. Data represent the mean ± SEM, n≥3. (H,I) Western blot analysis of ICAM-1 expression in ear tissue of wild-type, Dcn−/−, Sdc1−/− and Dcn−/−/Sdc1−/−mice at 24h ± DTH elicitation. (H) Semiquantitative densitometric analysis of ICAM-1 normalized to total protein expression. Data represent the mean ± SEM. (n≥4, P < 0.05, ** P < 0.01) (I) Representative Western blot. (J) TNF-α ELISA of ear tissue extracts of wild-type, Dcn−/− and Dcn−/−/Sdc1−/− mice at 24 h +/− DTH elicitation. Data represent the mean ± SEM. (n≥4, * P < 0.05).