Fig. 2: Rasgrp1 is coexpressed with Il6, and Rasgrp1 silencing selectively inhibits IL-6 protein expression in an acute inflammatory response.

a–c Quantitative PCR (Q-PCR) analysis of Il6 and Rasgrp1 mRNA expression in peritoneal macrophages treated with different doses of lipopolysaccharide (LPS; 0, 1, 10 or 100 ng/ml) (a), polyinosinic-polycytidylic acid (poly(I:C); 0, 5, 20 or 80 μg/ml) (b), or CpG oligodeoxynucleotide (0, 1, 3 or 9 μg/ml) (c) for 6 h. d–f qPCR analysis of Il6 and Rasgrp1 mRNA expression in peritoneal macrophages treated with 100 ng/ml LPS (d), 10 μg/ml poly (I:C) (e), or 5 μg/ml CpG ODN (f) for the indicated times (hours). g qPCR analysis of Il6 and Rasgrp1 mRNA expression in intestinal epithelial cells isolated from dextran sulphate sodium (DSS)-treated mice on the indicated days. h qPCR analysis of Il6 and Rasgrp1 mRNA expression in organs from mice intraperitoneally injected with LPS (15 mg per kg body weight) for 6 h. i qPCR analysis of Rasgrp1 mRNA expression in peritoneal macrophages 48 h after transfection with Rasgrp1 short interfering RNA (siRNA). RasGRP1 protein was examined by Western blotting (embedded image). j–l qPCR analysis of Il6 mRNA expression in peritoneal macrophages transfected as described in (a) and treated 48 h later with 100 ng/ml LPS (j), 10 mg/ml poly (I:C) (k) or 5 mM of CpG ODN (l) for the indicated times (hours). m–o ELISA and quantification of IL-6 in the supernatant of macrophages treated as described in (j–l). The values plotted are the means ± SEM of three independent experiments in all figures, paired t test, two-sided, for m, *p = 0.0352, *p = 0.0112, *p = 0.0124 in sequence; for n, *p = 0.0445, *p = 0.0488, *p = 0.0394 in sequence, for o, *p = 0.0099, *p = 0.0258, *p = 0.049 in sequence. ns, not significant.