Fig. 2
Analysis of the putative palmitoylation site C248. a Topology model of TNF-R1 depicting all intracellular cysteine residues and functional domains. The mutated C248 is indicated in red. b TNF-R1C248S was expressed in TNF-R1 deficient (ΔTNF-R1) cells. WB analysis of total TNF-R1 expression. The upper panel shows TNF-R1. The third panel shows the FLAG-tag of the construct. Low MW TNF-R1 is indicated by filled arrowheads, high MW TNF-R1 is indicated by empty arrowheads. β Actin serves as loading control. c Analysis of TNF-R1 surface expression and internalization. Left histogram: wildtype TNF-R1 expressing cells, right histogram: C248S-TNF-R1 expressing cells. ΔTNF-R1 (black curve), 0 °C/surface TNF-R1 (green) or 30 min, 37 °C/internalized TNF-R1 (red). The shift of the curve/increase in fluorescence intensity is due to TNF-R1 internalization and accumulation in endosomes (TNF-receptosomes). The dashed line marks the medium wt surface expression in both histograms. One representative experiment is shown. Sample images for wt (upper panels) and C248S cells (lower panels) are shown below. The left panels show surface TNF-R1, the right panels show internalized TNF-R1. d Apoptosis induction in U937 (wt, ΔTNF-R1 and C248S) analyzed by quantification of nuclear DNA fragmentation. Representative untreated cell with intact nucleus (violet) (upper left panel) and 20 h TNF treated cell with fragmented nucleus (upper right panel). The diagram shows mean values of 3 experiments with up to 10,000 imaged cells. e Degradation of IκB was analyzed upon stimulation of wt and TNF-R1C248S expressing cells with TNF by WB. Actin serves as loading control. One representative experiment is shown. f AcylRAC from TNF-R1wt (left side) and TNF-R1C248S (right side) expressing cells. Input represents the total membrane fraction used for acylRAC. The WB was probed for TNF-R1 or actin as negative control. Low MW TNF-R1 is indicated by filled arrowheads, high MW TNF-R1 is indicated by empty arrowheads