Fig 1: Atsttrin specifically binds to TNFR and DR3 in TNFR super family.A. cDNA encoding Atsttrin was fused to the Gal4 DNA binding domain in the pDBLeu vector and cDNAs encoding the extracellular portions of 28 TNFRSF members were fused to the Gal4 activation domain in the pPC86 vector. Selected plasmids were co-transformed into yeast strain MAV203; X-Gal assay was performed to determine β-galactosidase phenotype. B. Quantitative assay for β-galactosidase (β-gal) activity in liquid cultures of the interactions between Atsttrin and TNFR superfamily members were determined using o-nitropenyl-β-D-galactopyranoside (ONPG) as a substrate. For each strain, three independent colonies were analyzed and triplicate samples for each colony. pPC86 empty vector was used as a negative control. ***p<0.001 versus negative control.
Fig 2: The first three CRDs of DR3 extracellular portion are required for binding to Atsttrin. A. Schematic diagram of deletion constructs of DR3 extracellular portion used to map those fragments that bind to Atsttrin. Numbers refer to amino acid residues in the DR3. Interactions between Atsttrin and DR3 derivatives are summarized and indicated by “+” or “−”. B. β-Galactosidase activity was used to test interaction between various compositions of extracellular domains of DR3, as indicated, and Atsttrin. Two independent yeast transformants for each pair of plasmids were transferred onto a nitrocellulose membrane and the β-galactosidase activity was determined.
Fig 3: Atsttrin inhibits the binding of TL1A to DR3, similar to its inhibition of TNFa/ TNFR2 interaction.ELISA plate was coated with 100(A) or TL1A (B), and then 100 ng of TNFR2 or DR3 was added to each well respectively, in the presence of various Atsttrin, the bound TNFR2 or DR3 was detected by anti-TNFR2 or anti-DR3 antibody, followed by a secondary antibody conjugated with horseradish peroxidase.
Fig 4: Atsttrin directly binds to TNFR2 and DR3, examined by solid phase binding assay.A. SDS-PAGE analysis of purified Atsttrin. Atsttrin was indicated by an arrow. B and C. Solid phase binding assay. Various dose of Atsttrin was coated to ELISA plate, biotinylated TNFR2 (B), DR3 (C) or BSA (D) was then added to each well, bound protein was detected by adding avidin-HRP to each well and the absorbance was measured at OD 450 nm.
Supplier Page from R&D Systems, a Bio-Techne Brand for DR3/TNFRSF25 Fc Chimera Protein
Available conjugates: Sizes Available: 50 ug