TIRAP Functions as an Adaptor Protein for RAGE 10 mg/ml PMB, 5 mg/ml rexTLR2 and 5 mg/ml rexTLR4 was used to block activation of TLR2 and TLR4 by ligands. proteins, TIRAP, MyD88 and TRAM, were tagged with N- terminal Myc and C-terminal HA. Western Blot Analysis and Immunoprecipitation Western blot analysis was performed under conventional conditions. Antibodies used were as follows: mouse anti-HA tag (clone 6E2), mouse anti-Myc tag (clone 9B11), anti-human IRF-3; rabbit anti-human RAGE (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human phospho-IRF-3 (Ser396), rabbit anti-human p38, rabbit anti-human phospho-p38 (Thr180/ Tyr182), rabbit anti-human SAPK/JNK, mouse anti-human phospho-SAPK/JNK (Thr183/Tyr185), rabbit anti-human Akt, rabbit anti-human phospho-Akt (Ser473), rabbit anti-human IKKa, rabbit anti-human IKKa/b (Ser176/180), rabbit anti- human IRAK4, rabbit anti-human NFkB (p65), and mouse anti- human cleaved caspase-8 (Asp384) antibodies (Cell Signaling Technology, Beverly, MA); rabbit anti-His tag and rabbit anti- human MyD88 antibodies (MBL, Nagoya, Japan); rabbit anti- human PKCa, rabbit anti-human PKCe, rabbit anti-human PKCf, and rabbit anti-human PKCm antibodies (Santa Cruz Biotechnology); rabbit anti-human DIAPH1 (Dia-1), rabbit anti- human TIRAP, and mouse anti-TRAM antibodies (Abcam, Cambridge, MA); and mouse anti-human tubulin antibody (Sigma-Aldrich). Rabbit anti-human RAGE antibody was bioti- nylated using a Biotin Labeling Kit-SH (Dojindo Molecular Technologies, Rockville, MD) to recover antibody-free RAGE after immunoprecipitation using streptavidin-agarose (Figures. 2D, 3A, S3E and S3F). The second antibody was horseradish- peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (Cell Signaling Technology). Positive signals were detected by a chemiluminescence system (ECL plus, GE Healthcare Bio- Sciences, Piscataway, NJ). Rabbit anti-human IL-6 antibody (Abcam) was used for immunostaining. Monoclonal Anti-HA (clone HA-7) tag-agarose (Sigma-Aldrich), monoclonal Anti-His tag (clone 2D8) and monoclonal anti-Myc tag (clone 1G4) agaroses (MBL), and streptavidin agarose (Invitrogen) were used for the co-immunoprecipitation experi- ments. Northern Blot Analysis Ten micrograms of total RNA isolated by the acid guanidinium thiocyanate/phenol-chloroform method was electrophoresed in a 1% agarose gel and transferred to a Nytran Plus nylon membrane (GE Healthcare Bio-Sciences). Entire coding regions of human IL- 6, IL-8, TNFa, IFNb, and GAPDH genes were used as probes for Northern blot analysis. Plasmid Constructs Human cDNAs, S100A12, HMGB1, and cytoplasmic domain of RAGE (RAGE(Cyt), 364-404 aa) were cloned into pGEX vector series (GE Healthcare Bio-Sciences), i.e., S100A12 into pGEX-6P3, HMGB1 and RAGE(Cyt) into pGEX-6P1. We made a mammalian expression vector by modifying the pIDT-SMART cloning vector (Integrated Device Technology, San Jose, CA) using CMV promoter-intron (CMVi) and a part of the HTLV type 1 LTR (RU5’) to express even shorter cargo cDNAs at very high levels. Various combinations of tags at either the N-terminal (6His-2HA) or C-terminal (Myc-HA-Flag-6His) side of RAGE and its variant cDNAs were prepared. Since RAGE is a single-pass type I membrane protein, the tag sequence was put between the N-terminal region (1–31 aa) including the signal sequence (1–23 aa) and the remaining part of RAGE cDNA when constructing N-terminally tagged vectors (Figure. S1F). PKC isoforms were tagged with C-terminal HA, and TLR adaptor Recombinant Protein Preparation Dimerized human S100A11 was prepared as described previously [15]. Purification of the bacterially expressed GST- fusion proteins was performed under conventional conditions. GST was cleaved with PreScission protease (GE Healthcare Bio- Sciences) when necessary. Endotoxin (LPS and b-glucans) contaminated in the purified recombinant proteins were deter- mined by a Limulus Amebocyte Lysate (LAL) assay using a Limulus test kit (Seikagaku Corporation, Tokyo, Japan). Detected amounts were as follows: GST, 0.013 EU/mg; HMGB1, 0.017 EU/mg; S100A11, 0.007 EU/mg; S100A12, 0.008 EU/mg; AGE- BSA (Calbiochem), 0.001 EU/mg; and HMGB1 (R&D), 0.001 EU/mg. In vitro Kinase Assay In vitro kinase reaction was performed using active type of recombinant kinases (50 ng) and purified GST-RAGE(Cyt) (1 mg) or HEK293-derived RAGE(Cyt) purified by anti-6His tag antibody agarose (MBL) in the presence of [c-32P] ATP (HAS, Budapest, Hungary). To confirm activation of PKCf in cells, biotinylated-PKCf–specific substrate (Santa Cruz Biotechnology) was incubated with cell extracts, and then the substrate peptide was trapped on a streptavidin-coated plate (BD Biosciences Bedford, MA) and radioactivity was determined. Assay for Activated NFkB and Rac1 Pull-down assay of NFkB transcription factor bound to the NFkB-responsive element (NFkB RE) was performed under conditions reported previously [15]. A biotinylated 22-bp NFkB RE oligo DNA was prepared using a forward primer (59-biotin- AGTTGAGGGGACTTTCCCAGGC -39) and a reverse primer (39-TCAACTCCCCTGAAAGGGTCCG-biotin-59), incubated with whole cell extracts, and pulled down using a streptavidine agarose (Invitrogen). Bound NFkB (p65) was determined by Western blot analysis. GTP-bound form of Rac1 was determined using a Rac1 Activation Assay Kit (MILLIPORE). RNA Interference Validated siRNAs for human PKCa (ID No. s11092) and for human PKCf (ID No. s11129) and control siRNA (Silencer Negative control 2 siRNA) were purchased from Ambion (Austin, TX). Human PKCe (siGENOME SMART pool M-004653-02- 0005), human PKCm (siGENOME SMART pool M-005028-02- 0005), human RAGE (siGENOME SMART pool M-003625-02- 0005) and mouse TIRAP (siGENOME SMART pool M-049112- 00-0005) siRNAs were purchased from Thermo Scientific Dharmacon (Lafayette, CO). siRNAs were transfected using Lipofectamin RNAiMAX reagent (Invitrogen). Supporting Information Figure S1 Phosphorylation of RAGE and identification of a responsible kinase. (A and B) Phosphorylation of full- length RAGE (wt) but not cytoplasmic domain-deleted variant (DCyt). HEK293 cells were transfected with plasmid constructs of RAGE-Myc-HA-Flag-6His (wt, closed arrowhead; DCyt, open arrowhead), and the cells were then treated with 0,100 ng/ml of recombinant S100A11 protein for 1 h (A: The results were confirmed by an independent and four related experiments.) or with 10 ng/ml of the protein for indicated periods (B: The results were confirmed by an independent and four related experiments.). PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23132