Figure 1 - Functional analysis of Mal Ser180 and Mal Leu180.

(a) We assayed TLR2 signaling by measuring degradation of IB induced by Malp2. Top: wild-type MEFs stimulated with 5 nM Malp2. Second row: Malp2-stimulated Tirap knockout cells. Third row: Malp2-stimulated Tirap knockout cells transfected with Tirap Ser180. Fourth row: Malp2-stimulated Tirap knockout cells transfected with Tirap Leu180. Samples were immunoblotted with an antibody to IB. We used -actin as a loading control. Results shown are representative of three separate experiments. (b) Wild-type MEFs or Tirap knockout MEFs (transfected with empty vector or plasmids encoding Mal Ser180 or Mal Leu180) were treated with LPS (1 g/ml) or Malp2 (5 nM). After 24 h, supernatants were removed and assayed for IL-6 by ELISA (mean s.e.m. of triplicate experiments; we obtained similar results in two additional experiments). Expression levels of Mal Ser180 and Mal Leu180 (untreated or treated with LPS or Malp2 for 24 h) are shown in the lower panel. (c) Tirap knockout MEFs were transfected with 100 ng empty vector or with plasmids encoding Mal Ser180 or Mal Leu180 (50 ng of each, supplemented to 100 ng with 50 ng of empty vector) or a combination of plasmids encoding Mal Ser180 and Mal Leu180 (50 ng of each) in combination with a plasmid containing an NF-B-linked luciferase reporter gene (80 ng per well) and a Renilla luciferase reporter gene (40 ng) as an internal control. Cell lysates were prepared as previously described⁴. Results shown are mean + s.d. from triplicate experiments.