Fig. 5

tmTNF-α Ab facilitates LPS-induced TLR4 internalization and degradation. THP-1-derived macrophages were stimulated with 100 ng/ml LPS and 2 μg/ml tmTNF-α mAb or isotype IgG for indicated time points. a TLR4 expression on the cell surface was evaluated by flow cytometry. Representative images of FCM on the left, and quantitative data on the right. b Western blot analysis of TLR4 expression after stimulation for 1 h. THP-1-derived and Raw264.7 macrophages were stimulated with 100 ng/ml LPS, combined with 2 μg/ml tmTNF-α mAb or tmTNF-α pAb, respectively. Isotype antibody IgG or normal serum IgG served as a control. c Representative images of western blot analysis of TLR4 expression at 12 h after stimulation (upper) and their quantitative data (lower). d Relative levels of TLR4 mRNA were assessed by real-time PCR 4 h after stimulation. e THP-1-derived macrophages were stimulated with LPS and tmTNF-α mAb for indicated time points in the presence of 10 μg/ml cycloheximide. Representative images of western blot analysis of TLR4 expression (upper) and their quantitative data (lower). f THP-1-derived macrophages were treated for 4 h with 10 μM MG132 prior to the stimulation with LPS and tmTNF-α mAb for 12 h. Representative images of western blot analysis of TLR4 expression (upper) and their quantitative data (lower). g Western blot analysis of Triad3A expression in THP-1-derived or Raw264.7 macrophages stimulated with LPS and tmTNF-α mAb or pAb for 12 h, respectively. h Representative images of IP/western blot analysis of Triad3A recruited to TLR4 in THP-1-derived macrophages stimulated with LPS and tmTNF-α mAb for indicated time points. All quantitative data are presented as means ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001