Figure 6.
SM-induced necroptosis is TNF-R dependent. Bone marrow macrophages were derived from WT and TNFR1/2−/− mice and treated for 24 h with SM (5 μM) or SM/necrostatin. (a) The viability of cells after SM treatment was measured using MTT assay. Similar results were observed in three repeated experiments performed in triplicate. (b) Bone marrow macrophages from WT and TNFR1/2−/− mice were exposed to SM +/− Nec for varying time intervals as indicated. Cell lysates were then examined for Rip1 and Rip3 expression via western blot. (c) Bone-marrow-derived from WT macrophages were grown on cover slips in six-well plates and exposed to SM for 4 h. After staining with antibodies for TNF-R (blue), Rip1 (red) and Rip3 (green), cells were examined via fluorescent confocal microscopy. Images show an extended focus image composed of 10 overlaid z-stack images. (d) WT and TNFR1/2−/− macrophages were treated with SM +/− Nec for varying time as shown and lysed using RIPA buffer. Co-immunoprecipitation was then performed using Protein-G sepharose beads and an antibody targeting TNF-R1