FIGURE 7.

TRAF6 methylation correlates with TLR responses in humans. A, cytosolic and nuclear extracts were prepared from normal human liver tissue (liver transplant donor biopsy samples). Western blots were performed for PRMT1 and JMJD6 in cytosolic fractions and for NF-κB p65 in nuclear fractions. Data points represent values for individual liver specimens. n = 20. B, human peripheral blood monocytes were differentiated into macrophages and treated with or without AMI-1 for 16 h. Left panel, relative mRNA of TNFα in individual human macrophage preparations before (C) and after exposure to AMI-1. Right panel, -fold increase in TNFα mRNA 1 h after LPS exposure. Cells were pretreated with AMI-1 as indicated. Individual patient samples are connected by lines, n = 10. C, TRAF6 methylation was measured by ELISA as described under “Experimental Procedures.” Western blots were performed for JMJD6 protein concentration in cytosolic fractions from individual HBDM preparations. PRMT1 was measured using ELISA. n = 26. D, basal mRNA levels of TNFα and IL6 are plotted against relative TRAF6 methylation in individual HBDM preparations. E, -fold increase in IL6 mRNA levels is plotted against relative TLR4 expression or relative TRAF6 methylation in individual HBDM preparations. n = 26. Dependence of LPS-induced mRNA increases on TLR4 expression and TRAF6 methylation (6Tme) were statistically significant by multivariate linear regression, p = 0.0005 and 0.0017, respectively.