Fig. 4: PP6 promotes the activation of RIPK1. | Cell Death & Disease

Fig. 4: PP6 promotes the activation of RIPK1.

From: PP6 negatively modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL to promote TNFα-mediated cell death

Fig. 4

A–C PP6+/+ or PP6−/− MEFs were pretreated with Nec-1s for 1 h and then treated with TNFα/5Z-7, TNFα/zVAD/5Z-7, or TNFα/zVAD/CHX for indicated periods of time. Cells were lysed with RIPA buffer and analyzed by western blotting with indicated antibodies. D, E Control vector, WT-PP6 or D84N-PP6 reconstituted PP6−/− MEFs were treated with TNFα/zVAD/5Z-7 or TNFα/5Z-7 for indicated periods of time. Cells were lysed with NP-40 buffer and immunoprecipitated with anti-RIPK3 or anti-FADD antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. F c-FLIP−/− sgGFP or c-FLIP−/− sgPP6 MEFs were pretreated with Nec-1s for 1 h and then treated with TNFα/5Z-7 or TNFα/zVAD for indicated periods of time. G, H c-FLIP−/− sgGFP or c-FLIP−/− sgPP6 MEFs were treated with TNFα/5Z-7 or TNFα/zVAD/5Z-7 for indicated periods of time and then lysed with RIPA buffer. The lysates were analyzed by western blotting with indicated antibodies. Concentrations of reagents used: TNFα (T), 50 ng/ml; CHX (C), 2 μg/ml; Nec-1s, 10 μM; zVAD (Z), 50 μM; 5Z-7, 300 nM. The cell death in (F) was measured by CellTiter-Glo assay. Data represent mean ± SD of three independent experiments (Student’s t test ***P < 0.001; ****P < 0.0001).

Back to article page