Figure S5 Behavior of adaptor proteins and signal transduction upon S100A8/A9 stimulation. (A) Intracellu- lar signal transduction triggered by ligands in 293-hMD2-CD14 cells overexpressed 6His-2HA-RAGE and TLR4-Myc under same conditions described in Fig. S4A. The transfected cells were treated with control BSA (100 mg/ml), LBP-LPS (100 ng/ml each, preincubated for 2 h), AGE (100 mg/ml), HMGB1-LPS (100 ng/ ml each, preincubated for 2 h), and S100A8/A9 (100 ng/ml each) for 30 min. Open triangle, TLR4; closed triangle, RAGE. The results were confirmed by two independent and three related experiments. (B) Co-precipitation of endogenous adaptor proteins with full-length 6His-2HA-RAGE (wt) and TLR4 (wt)-Myc transfected into 293-hMD2-CD14 cells were performed under the conditions similar to those described in Fig. 4A. Cell extracts were pulled down with anti-Myc + anti-HA, anti-Myc, or anti-HA agarose and immunoblotted with indicated antibodies. 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