Figure 6. RIPK1 represses LPS-induced RIPK3 activity.

(a) WT BMDM were either pre-treated for 30 min with Nec-1 (50 μM), or cultured with Nec-1 in the final 30 min of priming with LPS (20 ng ml⁻¹) for 3 h. Cp.A (500 nM) was added, as indicated, and cells were cultured for 6 h. Supernatants were assayed for IL-1β. n=3 mice per group, mean+s.e.m. One of two experiments. (b) WT and Ripk1^−/− FLM, and WT and Ripk3^−/− BMDM were primed with LPS for 3 h and stimulated with Cp.A (500 nM) or Alum (300 μg ml⁻¹) for a further 6 h, and supernatants and lysates were assayed by immunoblot. Full-size immunoblots are presented in Supplementary Fig. 14. (c–e) WT, Ripk1^+/− and Ripk1^−/− FLDM were treated with glyburide for 20 min and then stimulated with LPS (20 ng ml⁻¹) for 6–8 h. Data shows three to five embryos of each genotype. (c,d) Supernatants were assayed for (c) IL-1β and (d) TNF production. Data symbols represent individual mice from three experiments. (e) Cell lysates and supernatants were blotted for caspase-1 cleavage. n=2 individual mice (numbered). Full-size immunoblots are presented in Supplementary Fig. 14. (f) WT Ripk1^+/+, Ripk1^−/−, Ripk3^−/− and Ripk1^−/−Ripk3^−/− FLDM were labelled with cell tracker green (green) and cultured with LPS (20 ng ml⁻¹) for 1 h, prior to stimulation with Cp.A, as indicated, and PI addition (red). Cells were imaged from 2h post-LPS addition every 30 min for 14 h. Supplementary Figure 4g shows additional treatments (LPS/Cp.A/QVD) used in this experiment. Representative images of one of three experiments. *NS, non-specific band.