Fig. 3
Phospho-Ser25 regulates the immune response against Yersinia. a Wild-type MEFs were pretreated with the indicated compounds for 30 min before stimulation with 1 µg/ml hTNF for the indicated duration. pS25 RIPK1 was then immunoprecipitated and treated with USP2 and λ phosphatase (PPase) post-IP when indicated. b–f Ripk1+/+ and Ripk1S25D/S25D BMDMs (b, e–f) or MEFs (c, d) were pretreated with the indicated compounds for 30 min before stimulation with hTNF (10 pg/ml for BMDMs and 100 pg/ml for MEFs) (b–d) or 50 ng/ml LPS for 6 h (e) or 4 h (f). Activation of cytosolic proteins was monitored by immunoblot (d) and cell death was measured in function of time (b–c, e–f). (g) Ripk1+/+ and Ripk1S25D/S25D BMDMs were infected with wild-type Y. enterocolitica (Ye) or with the YopP-negative mutant Ye.ΔP in presence or absence of Nec-1s. Cell death was quantified by SytoxGreen staining 4 h post-infection. h–i Ripk1+/+, Ripk1S25D/S25D and Ripk1K45A/K45A BMDMs were pretreated, or not, for 1 h with 5 µM IKK inhibitor BMS-345541 (IKKi), then infected with wild-type Y. pseudotuberculosis (Yp) or the YopJ-negative mutant Yp.ΔJ. Cell death was measured by LDH 6 h post-infection (h) and cytosolic RIPK1 activation was monitored by immunoblotting for pS166 RIPK1 (i). Cell death data are presented as mean ± SEM three independent experiments (c). BMDMs results were obtained with cells isolated from three (n = 3) (b, e–h) different mice of each genotype. Statistical significance for the cell death assays was determined using two-way ANOVA followed by a Tukey (b, c, h) or Sidak (g) post-hoc test. J BM chimeras were generated by reconstituting lethally irradiated congenic hosts with BM from either Ripk1+/+, Ripk1S25D/S25D, or Ripk1K45A/K45A mice. k–m BM chimeric mice were infected with 1–2 108 CFUs Y. pseudotuberculosis by oral gavage. Liver (k) (Ripk1+/+ n = 16, Ripk1S25D/S25D n = 13, Ripk1K45A/K45A n = 14) and spleen (l) (Ripk1+/+ n = 16, Ripk1S25D/S25D n = 13, Ripk1K45A/K45A n = 14) bacterial burdens were measured on day 5 post-infection and survival was recorded during two weeks (m) (Ripk1+/+ n = 6, Ripk1S25D/S25D n = 6, Ripk1K45A/K45A n = 6). Statistical significance for the bacterial burdens was determined using a Mann–Whitney test. Significance between samples is indicated in the figures as follows: ∗∗p < 0.01; ∗∗∗p < 0.001. Immunoblots are representative of two (d–i) independent experiments
