Mutations in TNFR1 prevent formation of forming soluble versions of the receptor. 293T cells were transiently transfected with the indicated TNFR1-YFP expression vectors without (A) or with (B) wild-type (WT) TNFR1-CFP. Medium was changed to serum-free medium and the cells were allowed to produce soluble TNFR1. Supernatants (Sup) and whole-cell lysates (WCL) were collected after 24 hours and analyzed by SDS-PAGE and immunoblotting with anti-TNFR1. eTFNR1 denotes exosome-associated full-length TNFR1; sTNFR1, soluble cleaved TNFR1 encoding the extracellular domain only.