Supplementary Materials and Methods

Expression and purification ofGrB,GrB-TWEAKand GrB-Fc-IT4.

Illustrations of the fusion constructs GrB,GrB-TWEAK, and GrB-Fc-IT4 are shown in Fig. 1A. Proteins were expressed in HEK-293T cells. Transient transfection of the pSecTag-GrB, pSecTag-GrB-TWEAK or pSecTag-GrB-Fc-IT4 expression vectors with Polyethylenimine (PEI) (1 µg/µl) was performed overnight at a 1:3 ratio of total DNA (µg) to PEI (µg), followed by incubation of cells in serum-free DMEM media for 72 h. Following dialysis of the conditioned media against 20 mMTris/300 mMNaCl, pH 7.6, recombinant proteins were purified using immobilized metal affinity chromatography (Ni2+-IMAC)as described elsewhere(1). Briefly, the conditioned media was loaded onto columns (2.5-cm internal diameter × 10 cm) containing nickel-charged IMAC resin. After washing with 20 mmol/L Tris-HCl (pH 7.6), 300 mmol/L NaCl, 5mmol/L imidazole, the bound protein was eluted with 20 mmol/L Tris-HCl (pH 7.6), 300 mmol/L NaCl, 200 mmol/L imidazole. Fractions containing fusion protein were dialyzed against 20 mmol/L Tris-HCl (pH 7.6)/150 mmol/L NaCl buffer, followed by digestion with recombinant enterokinase (15 units/mg for 48 h).

To further concentratethe fusion proteins, the protein (in 20 mmol/L Tris-HCl (pH 7.6) and 150 mmol/L NaCl) was loaded onto a small column containing 1 ml SP-Sepharose resin (GE Healthcare Biosciences, Pittsburgh, PA,). After washing with 20mmol/L Tris-HCl (pH 7.6) and 150 mmol/L NaCl, the protein was eluted using 20mMTris-HCl, 2 mol/L NaCl (pH 7.6). The purified protein was then dialyzed and stored in sterile PBS at -20C.

Surface plasmon resonance assay

Binding of TWEAK,GrB-TWEAK and GrB-Fc-IT4 to immobilized, recombinant Fn14 extracellular domain (Cell Sciences) was measured using a BIACore 3000 instrument aspreviously described(2). The binding to a blank cell (nonspecific binding) was subtracted from the sensorgram.

Reference List

(1) Zhou H, Liu Y, Cheung LH, Kim S, Zhang W, Mohamedali KA, et al. Characterization and mechanistic studies of a novel melanoma-targeting construct containing IkappaBa for specific inhibition of nuclear factor-kappaB activity. Neoplasia 2010;12:766-77.

(2) Brown SA, Hanscom HN, Vu H, Brew SA, Winkles JA. TWEAK binding to the Fn14 cysteine-rich domain depends on charged residues located in both the A1 and D2 modules. Biochem J 2006;397:297-304.

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