Mutant TNFR1 do not functionally associate with wild-type TNFR1, but self-associate through intermolecular disulfide bonds. (A) Jurkat 4E3 cells were transfected with the indicated ΔCD TNFR1-YFP expression vectors and treated with TNFα. Cell viability was then determined by forward scatter and PI uptake using flow cytometry. (B-C) FRET analysis of TNFR1 interactions. 293T cells were transfected with TNFR1-CFP and -YFP fusion proteins and analyzed by flow cytometry. In the top set of plots, wild-type (WT) TNFR1-CFP was cotransfected with WT-TNFR1 or Fas-YFP. The dot plot shows the CFP⁺YFP⁺ population gating used for FRET analysis in all samples. The bottom set of plots depicts association between CFP-tagged mutant receptors and WT (left) or mutant (right) YFP-tagged receptors. (C) Percentage of FRET-positive cells from all WT and mutant combinations tested, analyzed as in panel B. (D) 293T cells were transiently transfected with ΔCD TNFR1-YFP fusion proteins, and whole-cell lysates were run on a polyacrylamide gel in nonreducing (top) or reducing (bottom) conditions. The proteins were then immunoblotted using anti-TNFR1.