Fig. 6: EGLN1 hydroxylates IRF3 at proline 10.
a The hydroxylated residue in IRF3 was identified by mass spectrometry analysis. b Sequence alignment of partial IRF3 (1–20 amino acids). c HEK293T were transfected with the Flag empty vector (EV), Flag-IRF3 or Flag-IRF3-P10A, followed by immunoprecipitation and immunoblotting with the indicated antibodies. d Cell lysates from IRF3+/+ and IRF3−/− H1299 were extracted, followed by immunoprecipitation and immunoblotting with the indicated antibodies. e EGLN1−/− H1299 were transfected with Flag-IRF3-WT or Flag-IRF3-P10A, together with the HA empty vector (EV), HA-EGLN1-WT (WT), HA-EGLN1-H313A (H313A), followed by immunoprecipitation and immunoblotting with the indicated antibodies. f Cell lysates from EGLN1+/+ and EGLN1−/− H1299 were extracted, followed by immunoprecipitation and immunoblotting with the indicated antibodies. g Cell lysates from THP-1 under normoxia (21% O2) or hypoxia (1% O2) for 12 h were extracted, followed by immunoprecipitation and immunoblotting with the indicated antibodies. h Cell lysates from THP-1 treated with DMSO (vehicle control) or DMOG (1 mM) for 6 h were extracted, followed by immunoprecipitation and immunoblotting with the indicated antibodies. i Cell lysates from H1299 infected with VSV virus for the indicated time were extracted, followed by immunoprecipitation and immunoblotting with the indicated antibodies. j Quantitation of the intensity of hydroxylated IRF3/total IRF3 protein in (i). k Bacterially expressed HIF1α (400-575aa) (GST tag has been removed using thrombin) incubated with bacterially expressed GST or bacterially expressed GST-EGLN1 for hydroxylation reaction and then detected by immunoblotting with anti-HIF1α-P564-OH antibody. l In vitro hydroxylation assay to detect bacterially expressed IRF3 hydroxylated by bacterially expressed GST-EGLN1. m Bacterially expressed IRF3 (GST tag has been removed using thrombin) was incubated with bacterially expressed GST-EGLN1 for hydroxylation reaction and then identified by mass spectrometry analysis. Hydroxylated IRF3-P10 is indicated by the red arrow, and the percentage of IRF3 hydroxylation is 10.34%. EV empty vector, IP immunoprecipitation, TCL total cell lysates. Data in (c–i, k, l) are representative from three independent experiments. See also Supplementary Fig. 11. Source data are provided as a Source data file.
