Extended Data Fig. 5: The IRF6 phosphomimetic knock-in mouse demonstrates that phosphorylation of IRF6 at Ser413 and Ser424 is essential to prime it for activation.
From: The RIPK4–IRF6 signalling axis safeguards epidermal differentiation and barrier function
a, Western blots of wild-type, Irf6+/S413A,S424A and Irf6S413A,S424A/S413A,S424A E18.5 skin. Representative of three independent experiments. b, E18.5 skin sections (n = 3 wild type and n = 3 Irf6S413A,S424A/S413A,S424A) stained with H&E or antibodies against K10, K14 and Ki67. Scale bars, 50 μm. c, E18.5 sections (n = 3 wild type and n = 3 Irf6S413A,S424A/S413A,S424A) stained with H&E, showing fusion of the stratified squamous portion of the stomach in the Irf6S413A,S424A/S413A.S424A embryos. Scale bars, 100 μm. d, Graph indicates activation of an IRF-responsive luciferase reporter gene at 24 h after transfection of 293T cells with the indicated IRF6 and RIPK4 constructs. IRF6 activity is displayed as fold activity over reporter only. Data are mean ± s.d. (n = 5). IRF6(R84C) is a DNA-binding mutant, and represents a negative control in this experiment. Unpaired two-tailed Fisher’s exact test with 95% confidence interval. e, Western blots of wild-type, Irf6S413E,S424E/S413E,S424E and Irf6−/− E18.5 skin. Representative of three independent experiments. f, E18.5 skin sections from wild-type, Irf6S413E,S424E/S413E,S424E, Irf6S413E,S424E/S413E,S424ERipk4D161N/D161N, Irf6S413E,S424E/S413E,S424ERipk4+/D161N and Irf6+/S413E,S424ERipk4D161N/D161N embryos (n = 3 each) stained with antibodies against K10, K14 and Ki67. Scale bars, 50 μm. g, E18.5 sections from wild-type, Irf6S413E,S424E/S413E,S424E, Irf6S413E,S424E/S413E,S424ERipk4D161N/D161N, Irf6S413E,S424E/S413E,S424ERipk4+/D161N and Irf6+/S413E,S424ERipk4D161N/D161N embryos (n = 3 each) stained with H&E, showing fusion of the squamous epithelium at the mouth and fusion of the tongue to the palate. Scale bars, 100 μm. h, Table showing the observed and expected numbers at E18.5 of offspring that were generated from intercrossing Ripk4+/D161NIrf6+/S413E,S424E mice. i, Table showing the observed and expected numbers at E18.5 of offspring that were generated from intercrossing Irf6+/S413E.S424E mice.
