Retention of TRAPS mutant TNFR1 in the ER. (A-B) HT1080 cells were cotransfected with YFP fusion proteins of full-length wild-type or mutant TNFR1 and CFP fusion proteins of the Golgi marker GalT or the ER marker Srβ. Images were acquired by confocal microscopy. YFP fluorescence is shown in green and CFP fluorescence in red. (C) TRAPS mutant TNFR1 localization is not altered by Brefeldin A. T50M TNFR1-YFP was cotransfected with GalT-CFP as in panel A and cells were treated with Brefeldin A prior to confocal microscopy. (D) FRAP was analyzed in COS-7 cells transfected with wild-type and mutant forms of TNFR1 with the cytoplasmic domain truncated. A small area of the cell was bleached with the laser on high power, and the return of fluorescence to that area over time was monitored. t_1/2 ± SD (n = 5) is shown for each condition.