Extended Data Fig. 8: pVHL suppresses RIPK1 activity.
(a-c) Cell lysates from primary human fibroblasts (a), RIPK1-/- HT-29 (b,c) cells infected with indicated expression vectors with (b) or without (c) DFO (200 μM) were immunoprecipitated. The immunocomplexes were immunoblotted. Data are mean ± s.e.m. of n = 3 independent experiments (c). (d-f) Cell lysates from 293 T cells transfected with indicated expression vectors with (e) or without (d) 10 μM Nec-1s, immunoprecipitated using anti-Flag (e), the reaction products of in vitro hydroxylation assays (f), incubated with or without 100 μM ATP, 50 μM Nec-1s or purified recombinant VHL and hydroxylated HIF1α peptide at 30 °C for 30 min. The samples were analysed by immunoblotting. (g) A sequence alignment of indicated proteins using the Clustal Omega program. (h) A schematic representation of various biotinylated synthetic RIPK1-derived peptides. (i) Cell lysates from 293 T cells transfected with indicated expression vectors for 24 h, were incubated with RIPK1-derived peptides for 4 h. The immunocomplexes were analysed by immunoblotting. (j) Coomassie staining of the bacterially purified recombinant pVHL/Elongin-C/Elongin-B complex. (k-l) A structural overlay of pVHL/Elongin-C/Elongin-B bound to either HIF1α or RIPK1. (m) Details of interaction between pVHL and HIF-1α (PDB: 4AJY). (n) 293 T cells were transfected with indicated expression vectors for 24 h. The levels of indicated proteins were determined by immunoblotting. (o-q) Cell lysates from Vhl KO MEFs (o), Vhl KO HT-29 cells (p), Cul2 knockdown HT-29 cells (q) were immunoblotted. (r-t) Cell lysates from VHL KO 293 T cells (r), MEFs (s), and 786-O cells (t) were immunoprecipitated with anti-RIPK1 antibody, and immunoblotted. (u-v) Vhl KO MEFs reconstituted with indicated expression vectors exposed to hypoxia. Cell death was measured by PI uptake assay. Data are mean ± s.d. of n = 3 biological independent samples. Two-way ANOVA, post hoc Bonferroni’s test (u). Results are representative of three independent experiments. (a-f, i, j,n-t, v).
