TRAPS mutations render TNFR1 unable to bind TNFα. (A) Surface plasmon resonance analysis of TNFα binding. Fc-fusions of wild-type (WT), the indicated mutant TNFR1-Fc, and control DR5-CRD1-Fc were injected onto BIAcore chips coated with immobilized TNFα. Sensorgrams show resonance units (RU) over time. (B) In vitro pull-down of TNFα with Fc-fusion proteins. The indicated purified Fc-fusion proteins were mixed with protein-A Sepharose beads and tissue-culture supernatant containing soluble TNFα-FLAG. After washing, proteins were removed from the Sepharose and run on SDS-PAGE. Gels were stained with Coomassie blue. The final lane is a pull-down of supernatant alone (sup) with anti-FLAG beads to confirm presence of TNFα. Nonspecific bands are indicated by an asterisk. (C) Immunoprecipitation of TNFR1-GFP2 with TNFα-FLAG. Lysate from 293T cells transfected with wild-type or TRAPS mutant ΔCD GFP2-fusion protein expression vectors or a CTLA4-GFP2 control fusion vector were mixed with anti-FLAG Sepharose beads precoated with TNFα-FLAG. After washing, proteins were eluted from the beads and run on SDS-PAGE along with whole-cell lysates (WCL). Immunoblots were performed with anti-GFP–HRP.
