Mutant TNFR1 do not traffic to the plasma membrane. (A) 293T cells were transiently transfected with the indicated full-length wild-type (WT) or mutant TNFR1-YFP fusion constructs with N-terminal HA tags. Live or fixed and permeabilized cells were then stained with anti-HA followed by anti–mouse PE and analyzed by flow cytometry. The histograms are gated on the YFP⁺ cells that were live according to forward- and side-scatter characteristics. (B) 293T cells were transfected as with ΔCD FLAG-TNFR1-GFP² and stained as in panel A with anti-FLAG. The filled gray and black areas in panels A and B represent cells transfected with a YFP expression vector and stained as in panel A. (C) N-terminal HA-tagged WT TNFR1-CFP was cotransfected with wild-type or the indicated mutant N-terminal FLAG-tagged TNFR1-ΔCD-GFP². Cells were surface stained with anti-HA and anti-FLAG antibodies and analyzed by flow cytometry. Plots shown are gated on the CFP⁺GFP⁺ population.