Processing and subcellular distribution of sTNFR1-tm. (A) RBL cells expressing sTNFR1-tm were radiolabeled (pulse) for 1 hour and the radiolabel chased for up to 12 hours. At the indicated time points, 20 × 106 cells and incubation medium were withdrawn, immunoprecipitated, and analyzed as described in “Materials and methods.” The position of sTNFR1-tm is indicated with an arrow, and a processed form (that is, the secreted form) with a dotted line. Molecular weight markers are shown to the left in kDa. (B) Cells were biosynthetically radiolabeled for 1 hour (pulse), followed by a radiolabel chase for 2 hours. At the pulse and chase intervals, 100 × 106 cells were removed and homogenized, and the postnuclear supernatant was subcellularly fractionated by centrifugation in Percoll. Extraction, immunoprecipitation, and analysis were performed as described in “Materials and methods.” The densitometry data are shown for pulse (□) and chase (##) experiments as percent of total sTNFR1-tm and processed forms.
