Figure 2. Dab2 regulates LPS-induced TLR4/TRIF-dependent inflammatory response.

(a) Dab2 expression in the shLuc, shDab2-B and shDab2-C cells was determined by Western blotting using the anti-Dab2 (p96) antibody. The expression of β-actin was used as a control of equal protein. The relative expression level of Dab2 after normalization by the expression of β-actin is shown. The full Western blot and the corresponding positions of the molecular weight protein markers are presented in Supplementary Fig. S2. (b) The total RNAs obtained from the shLuc and shDab2-B cells with or without LPS treatment for 6 h were subject to microarray analysis. The 926 normalized genes that were differentially expressed by treatment with LPS (LL vs. LC, and DL vs. DC) or by knockdown of Dab2 (DC vs. LC, or DL vs. LL) with the Log₂ ratio >1.5 or <−1.5 are shown in the heat map. LC, shLuc cells without LPS treatment; LL, shLuc cells with LPS treatment; DC, shDab2-B cells without LPS treatment; and DL, shDab2-B cells with LPS treatment. (c,d) Significant gene ontology enrichment analysis related to biological process (c) and KEGG pathway (d) ordered according to –log (p-value) (e) The shLuc and shDab2-B cells were treated with LPS (100 ng/ml) for 3 and 6 h. The mRNA expression of pro-inflammatory mediators was determined by real-time RT-PCR and was normalized by the expression of gapdh. The levels of each cytokine mRNA expression for the shLuc cells without LPS treatment were arbitrarily set as 1. Data represent the mean ± SEM of three independent experiments. (f) The shLuc and shDab2-B cells were treated with LPS (100 ng/ml) for the indicated time. The culture medium was collected for ELISA assays to measure the secreted levels of the indicated pro-inflammatory mediators. Data represent the mean ± SEM of three independent experiments. (g) The shLuc, shDab2-B and shDab2-C cells were treated with LPS (100 ng/ml) for 6 h. Total RNAs were isolated from the indicated cell lines for real-time RT-PCR analyses of the indicated pro-inflammatory mediators. Data represent the mean ± SEM for the fold increase of the pro-inflammatory mediators mRNA in shDab2-B or shDab2-C cells when compared with the LPS-treated shLuc cells in a representative experiment. At least four independent experiments were performed. n.s., no significance. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.