. St, . Louis, and U. Mo, For western blot analysis of human proteins, anti-TRAIL-R2 antibody was purchased from Chemicon (Millipore Le Pont de Claix, France), anti-caspase-8 and anti-caspase-10 were from Medical & Biological Laboratories (Clinisciences Murine DR5 was revealed by western blot using the anti-mouse TRAIL-R2 antibody from Leinco Technologies Flow cytometry analysis for murine DR5 was performed using the MD5-1 antibody, with a PE-conjugated anti-armenian hamster secondary antibody from BD Biosciences. For human receptors, anti- TRAIL-R1 (wB-S26) and anti-TRAIL-R2 (B-D37) antibodies were provided by Gen-Probe (Diaclone The secondary antibody was an Alexa- 488-coupled goat anti-mouse from Molecular Probes Tunicamycin and swainsonine were from Sigma-Aldrich For apoptosis measurement, Crosslinking of TRAIL was achieved by incubating the ligand with anti-Flag M2 antibody for 30 min at 4 °C with mixing prior to cell treatment anti-FADD from Transduction Laboratories (BD Biosciences V (556422) and 7AAD (559925) were from BD Biosciences

. Plasmid-constructions, The parental murine full-length TRAIL-R and human TRAIL-R1 or TRAIL-R2 retroviral pMSCV vectors were obtained by subcloning using HindIII/XhoI from the pCR3-mTRAIL-R, pCR3-TRAIL-R1 and pCR3-hTRAIL-R2 vectors. 12,13 Chimeric mouse/human TRAIL receptor constructs were generated as follows. First, a modified version of the full-length hTRAIL-R2 harboring two restriction sites, BglII and BamHI, encompassing the transmembrane domain was obtained by synthesis from, ) and subcloned into the pMSCV retroviral vector using HindIII/XhoI to generate pMSCV-hTRAIL-R2-BglII-TM-BamHI

. Next, . Extracellular, . Intracellular-domains, I. Ecd, A. Respectively et al., were obtained by PCR from pCR3-mTRAIL-R using the following primer sets: mTRAIL-R (ECD) 5?-aaa aga tct, PCR products were digested using BglII/ApaI or HindIII/ BamHI, respectively, and digestion products were inserted

5. Bglii-tm-bamhi, 5. , 5. , 5. , 5. et al., ICD): referred to in the text as h/mTRAIL-R or m/ hTRAIL-R2. Human TRAIL-R1 N156A or N156Q and Mouse TRAIL receptor variants N99A, N122A, N150A mutants were created by routine site-directed mutagenesis from the pMSCV-mTRAIL-R vector using the following sets of primers, p.156

N. , 5. , and 5. Ag, 3? to generate pMSCV-hTRAIL-R1-N156A, pMSCV-hTRAIL-R1-N156Q, pMSCV-mTRAIL-R-N99A; pMSCV-mTRAIL-R-N122A and pMSCV-mTRAIL-R-N150A. Double and triple mutants were obtained as above through successive site-directed mutagenesis to generate pMSCV-mTRAIL-R-N99/122A; pMSCV-mTRAIL-R-N122/150A, pMSCV-mTRAIL-R- N99/150A and pMSCV-mTRAIL-R-N99/122/150A. The UL141-GFP fusion plasmid was generated as described, 5 and the UL141-GPI expression plasmid was generated by PCR amplification of the UL141 ectodomain, FIX strain 5 ) and cloning upstream of the sequence encoding for the addition of the TRAIL-R3 GPI linkage. 14 Sequences of all constructs were confirmed by sequencing

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