Fig. 2

TLR-2 is the direct binding receptor of HBeAg when activating macrophages. After pretreated with isotype IgG or blocking antibodies for TLR-2/3/4 (5 μg/ml) for 30 min, RAW264.7 macrophages were stimulated with HBeAg for 4 h. The expression and secretion of IL-6 were analyzed by qRT-PCR and ELISA (A). The siRNAs for TLR-2 or negative control were transfected into hMDM for 48 h, and then the cells were treated with HBeAg for 4 h. The expression of TLR-2 was tested by western blot assay (B), and the level of IL-6 (C) and TNF-α (D) were tested by ELISA. Mouse Kupffer cells were treated with DMSO or inhibitors of TLR-2 for 30 min, and then they were treated with HBeAg for 4 h. The level of IL-6 (E) and TNF-α (F) were tested by ELISA. Co-IP analyzed the direct binding between recombinant GST-HBeAg and recombinant extracellular segment of TLR-2 proteins in vitro (G, H). RAW264.7 macrophages were treated with GST-HBeAg (2 μg/ml) for 1 h, and then the cells were lysed. Cell lysis was incubated with GST antibody or isotype IgG overnight at 4°C, and the direct binding between endogenous TLR-2 and recombinant GST-HBeAg was analyzed by western blot (I). The siRNAs for TLR-1, TLR-6, or negative control were transfected into RAW264.7 macrophages for 48 h, and then the cells were treated with HBeAg for 4 h. The expression of TLR-1 or TLR-6 were tested by western blot assay respectively (J, M), and the corresponding levels of IL-6 (K, N) and TNF-α (L, O) were tested by ELISA respectively. The siRNAs for TLR-1, TLR-6, or negative control were transfected into PMA-pretreated THP-1 macrophages for 48 h, and then the cells were treated with HBeAg for 4 h. The expression of TLR-1 or TLR-6 were tested by western blot assay respectively (P, S), and the corresponding levels of IL-6 (Q, T) and TNF-α (R, U) were tested by ELISA respectively. *P < 0.05, **P < 0.01, ***P < 0.001