Tnfri

The cells or tissue biopsy samples were homogenized in a protein lysis buffer. A 50-μg protein sample was separated through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were immunoblotted with various primary antibodies, followed by incubation with the appropriate peroxidase-coupled secondary antibodies. Furthermore, the signal was visualized on an X-ray film after detecting with an enhanced chemiluminescent detection system. Antibodies to p21, p-p53 (phosphorylated at S15, S46, and S392), tumor necrosis factor receptor I (TNF RI), Fas-associated death domain protein (FADD), cellular inhibitor of apoptosis protein 1 (cIAP-1), hypoxia-inducible factor (HIF)-1α, and TNF-related apoptosis-inducing ligand receptor 1 (TRAIL R1) were purchased from R&D Systems.

DARPins have been described previously [19 (link), 21 ]. Two rounds of cell-based affinity selections were each performed on 10⁷ activated expanded Treg cells originating from seven donors using a DARPin phage display library containing up to 10⁹ binding members, as described previously [17 (link), 18 (link)]. The following recombinant human proteins were included as de-selection antigens: TCR α and β chains, CD5 (Cambridge Biosciences); CD69, CD3ε, and ITGB2 (Sino Biological); CD2 (Abcam); CD132, CD122, CD39, IL-10Rα, sCD4, CD109, IL-1R, IL-6Rα, IL-14Rα, IFN-γR1, CD45, CD38, TNFRI, 4-1BB, CD30, GITR, PD-1, B7-H1, CD44, CD25, CD27, and CD28 (R&DSystems). DARPins isolated from two rounds of selections were reformatted as either human IgG₁ Fc domain fusions or mouse IgG_2a Fc domain fusions and expressed in HEK293 cells (ATCC).