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. 2017 Jun 1;31(11):1162–1176. doi: 10.1101/gad.299776.117

Figure 3.

Figure 3.

SPATA2 is required for TNFα-induced apoptosis. (A,B) Immortalized Spata2+/+ MEFs and Spata2−/− MEFs were pretreated with 20 µM Nec-1s for 30 min and treated with 100 nM 5Z-7 and 50 ng/mL human TNFα for 12 h. Cell viability was measured by CellTiterGlo assay. (C) Immortalized Spata2+/+ MEFs and Spata2−/− MEFs were pretreated with 20 µM Nec-1s for 30 min and treated with 100 nM TPCA-1 and 50 ng/mL human TNFα for 12 h. Cell viability was measured by CellTiterGlo assay. (D) Immortalized Spata2−/− MEFs and Spata2+ MEFs were treated with 100 nM 5Z-7 and 100 ng/mL human TNFα for the indicated periods of time. The cell lysates were analyzed by immunoprecipitation using anti-p-S166 RIPK1. The immunocomplexes were analyzed by Western blotting. (E) Immortalized Spata2+/+ MEFs and Spata2−/− MEFs were treated with 100 nM 5Z-7 and 100 ng/mL human TNFα for the indicated periods of time. The cell lysates were analyzed by Western blotting using the indicated antibodies. (F) Immortalized Spata2−/− MEFs and Spata2+ MEFs were treated with 100 nM 5Z-7 and 100 ng/mL human TNFα for 12 h. Caspase-8 activity was measured according to the manufacturer's protocol. (G) Immortalized Spata2+/+ MEFs and Spata2−/− MEFs were pretreated with 20 µM Nec-1s and 25 nM SM-164 for 30 min and treated with 50 ng/mL human TNFα for 12 h. Cell viability was measured by CellTiterGlo assay. Error bars represent SEM from three technical replicates. Tubulin was used as a loading control.