Figure 5.
IFN-γ enhances the binding of RIPK1 to JAK1 to activate STAT1 in IECs. (A) HEK293T cells were cotransfected with a DNA plasmid expressing Flag-tagged JAK1 plus the plasmid expressing HA-tagged RIPK1 or RIPK3. Cell lysates were collected and immunoprecipitated with anti-Flag agarose. The Flag-JAK1 immunocomplex was analyzed by immunoblotting analysis. (B) HEK293T cells were cotransfected with a DNA plasmid expressing Flag-tagged RIPK1 plus the plasmid expressing HA-tagged JAK1 or the truncated form of JAK1 as indicated. Cell lysates were collected and immunoprecipitated with anti-Flag agarose. The Flag-RIPK1 immunocomplex was analyzed by immunoblotting analysis. (C) Intestinal crypt cells isolated from WT and Ripk3−/− B6 mice were treated with IFN-γ for 1 hour. Representative images of in situ PLA between JAK1 and RIPK1 (red). 4′,6-diamidino-2-phenylindole was shown (blue). Bar represents 5 μm. (D) Immunoblotting analysis of p-STAT1, c-myc-tagged STAT1, Flag tagged-RIPK1, HA tagged-RIPK3, and β-actin in HEK293T cells transfected with the indicated DNA plasmid(s). Quantification of p-STAT1 or STAT1 normalized to β-actin was shown under the band. (E) Intestinal crypt cells isolated from WT, Ripk3−/−, and Mlkl−/− B6 mice were treated with IFN-γ for 0.5 hour. Immunoblotting analysis of p-STAT1, STAT1, RIPK3, MLKL, and β-actin. Quantification of p-STAT1 or STAT1 normalized to β-actin was shown under the band. (F) Intestinal crypt cells isolated from WT and Ripk1K45A/K45A B6 mice were treated with IFN-γ for 0.5 hour. Immunoblotting analysis of p-STAT1, STAT1, and β-actin. Quantification of p-STAT1 or STAT1 normalized to β-actin was shown under the band. (G) Intestinal crypt cells isolated from WT, Ripk1K45A/K45A, and Ripk3−/− B6 mice were treated with IFN-γ for 0.5 hour. STAT1 binding to selected regions of the Cxcl9 and Cxcl10 promoters was determined by ChIP-qPCR. The amount of precipitated DNA was calculated as percent input. (H-I) Intestinal crypt cells isolated from WT, Ripk3−/−, Mlkl−/−, and Ripk1K45A/K45A B6 mice were treated with IFN-γ for 24 hours, and the culture medium was collected for ELISA measurement of CXCL9 protein (H), and for further experiment (I). The culture medium was then added into the bottom compartment of a transwell chamber and T cells isolated from WT B6 mice were plated into the upper compartment. After 6 hours, migration of T cells was assessed by transwell assay (I). Data shown are representative of 3 independent experiments. Data are shown as the mean ± SD. ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001. Multiple comparisons were evaluated by one-way ANOVA. ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunosorbent assay analysis; n.s., not significant; PLA, proximity ligation assay. SD, standard deviation.

IFN-γ enhances the binding of RIPK1 to JAK1 to activate STAT1 in IECs. (A) HEK293T cells were cotransfected with a DNA plasmid expressing Flag-tagged JAK1 plus the plasmid expressing HA-tagged RIPK1 or RIPK3. Cell lysates were collected and immunoprecipitated with anti-Flag agarose. The Flag-JAK1 immunocomplex was analyzed by immunoblotting analysis. (B) HEK293T cells were cotransfected with a DNA plasmid expressing Flag-tagged RIPK1 plus the plasmid expressing HA-tagged JAK1 or the truncated form of JAK1 as indicated. Cell lysates were collected and immunoprecipitated with anti-Flag agarose. The Flag-RIPK1 immunocomplex was analyzed by immunoblotting analysis. (C) Intestinal crypt cells isolated from WT and Ripk3−/− B6 mice were treated with IFN-γ for 1 hour. Representative images of in situ PLA between JAK1 and RIPK1 (red). 4′,6-diamidino-2-phenylindole was shown (blue). Bar represents 5 μm. (D) Immunoblotting analysis of p-STAT1, c-myc-tagged STAT1, Flag tagged-RIPK1, HA tagged-RIPK3, and β-actin in HEK293T cells transfected with the indicated DNA plasmid(s). Quantification of p-STAT1 or STAT1 normalized to β-actin was shown under the band. (E) Intestinal crypt cells isolated from WT, Ripk3−/−, and Mlkl−/− B6 mice were treated with IFN-γ for 0.5 hour. Immunoblotting analysis of p-STAT1, STAT1, RIPK3, MLKL, and β-actin. Quantification of p-STAT1 or STAT1 normalized to β-actin was shown under the band. (F) Intestinal crypt cells isolated from WT and Ripk1K45A/K45A B6 mice were treated with IFN-γ for 0.5 hour. Immunoblotting analysis of p-STAT1, STAT1, and β-actin. Quantification of p-STAT1 or STAT1 normalized to β-actin was shown under the band. (G) Intestinal crypt cells isolated from WT, Ripk1K45A/K45A, and Ripk3−/− B6 mice were treated with IFN-γ for 0.5 hour. STAT1 binding to selected regions of the Cxcl9 and Cxcl10 promoters was determined by ChIP-qPCR. The amount of precipitated DNA was calculated as percent input. (H-I) Intestinal crypt cells isolated from WT, Ripk3−/−, Mlkl−/−, and Ripk1K45A/K45A B6 mice were treated with IFN-γ for 24 hours, and the culture medium was collected for ELISA measurement of CXCL9 protein (H), and for further experiment (I). The culture medium was then added into the bottom compartment of a transwell chamber and T cells isolated from WT B6 mice were plated into the upper compartment. After 6 hours, migration of T cells was assessed by transwell assay (I). Data shown are representative of 3 independent experiments. Data are shown as the mean ± SD. ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001. Multiple comparisons were evaluated by one-way ANOVA. ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunosorbent assay analysis; n.s., not significant; PLA, proximity ligation assay. SD, standard deviation.

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