Fig. 4

AAV-mediated RIPK1 S321A mutant expression induces liver damage in vivo. a Phosphorylation of RIPK1 S321 in mice tissues after TNFα injection. 30 µg TNFα were delivered to mice (8-week-old male) through intraperitoneal injections. Tissues were collected at 30 and 60 min after the injection. RIPK1 was immunoprecipitated from tissue lysates by RIPK1 total antibody. b, c Liver damage in mice induced by AAV-mediated RIPK1 S321A mutant expression. AAVs carrying RIPK1 WT, S321A, S321E or GFP as a control were delivered through tail vein injection. Sixteen days after the AAV injection, the mice were treated with DMSO (D, n = 9) or Nec-1s (N, n = 8) through oral dosing. Four weeks after the AAV injection, plasma and liver samples were collected. ALT b and TNFα ELISA c assays were performed as manufacture’s instructions. d TUNEL staining for assessing liver damage induced by AAV-mediated RIPK1 expression. Liver sections were stained with TUNEL and Hoechst. Cells with co-localized TUNEL and Hoechst signals were counted as TUNEL-positive. e AAV-mediated expression of FLAG-RIPK1 variants in liver. The liver lysates were subjected to anti-FLAG immunoprecipitation to differentiate endogenous RIPK1 and ectopic FLAG-RIPK1. Relative intensity of cleaved (cl.) RIPK1 bands was quantified. Error bar, s.e.m. **, t-test P < 0.01, ***P < 0.001. Three independent repeats were included in each data point