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Dual-luciferase reporter assay. Dual-luciferase reporter assay was performed mainly as described previously [6].
Cross-linking of Proteins in vivo. 293T Cells were lysed in MI-773 Triton X-100-based lysis buffer for 1 h and the nuclear and cellular debris were cleared by centrifugation. The supernatants were then collected and treated with DMSO alone or disuccinimidyl suberate (DSS) in DMSO from a 10-fold stock solution to a final concentration of 1 mM. After incubating at 37 °C for 30 min, the cross-linker was quenched by the adding of 1 M Tris–HCl (pH7.5) to a final concentration of 20 mM at room temperature for 15 min. Samples were then solubilized in sample buffer (Bio-Rad), boiled and centrifuged at 13,200 rpm for 5 min.
Results
The NLS (aa 442–472) of RIP3 is essential for apoptosis
Fig. 1.
The NLS (aa 442–472) of RIP3 is essential for apoptosis. (A) Schematic diagrams of constructs used in this figure: pEGFP-C1/RIP3, pEGFP-C1/RIP3-NLS(aa 442–472), pEGFP-C1/RIP3ΔNLS (aa 442–472), pEGFP-C1/RIP3(I452A). Numbers indicate the position of amino acid in RIP3. (B) HeLa cells were transfected with the indicated plasmids. At 24 h post-transfection, the viability of cells was measured by counting GFP positive apoptotic cells characteristic of aberrant nuclei stained by Hoechst 33342 and the data were plotted as percent apoptosis. Values shown are means (±SD) of independent experiments in triplicate.