TIRAP Functions as an Adaptor Protein for RAGE Figure 4. Behavior of adaptor proteins and signal transduction upon differential stimulation of RAGE and TLR4. (A) Co-precipitation of endogenous adaptor proteins with full-length 6His-2HA-RAGE(wt) and TLR4(wt)-Myc transfected into 293-hMD2-CD14 cells. The cells were treated with control BSA (100 mg/ml), LBP-LPS (100 ng/ml each, preincubated for 2 h), AGE (100 mg/ml), and HMGB1-LPS (100 ng/ml each, preincubated for 2 h) for 30 min. Cell extracts were pulled down with anti-Myc + anti-HA, anti-Myc, or anti-HA agarose and immunoblotted with indicated antibodies. Open triangle, TLR4; closed triangle, RAGE. The results were confirmed by two independent and two related experiments. (B) Co-precipitation of endogenous adaptor proteins with RAGE-Myc-HA-Flag-6His and TLR4-Myc (w, wild-type; D, cytoplasmic domain-deleted variant). Experiments were PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23132