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. 2018 Oct 1;215(10):2567–2585. doi: 10.1084/jem.20180628

Figure 2.

Figure 2.

Impact of IRF9 Δex7 on IFN receptor-proximal signaling. (A) qRT-PCR measuring of IRF9 mRNA levels in PBMCs from the patient, her mother, and a healthy control with two probes—one probe spanning intron 7, and a second probe spanning intron 1. Representative results of four independent experiments are shown. (B) Top: WB of endogenous IRF9 in patient F-SV40 cells; GAPDH was used as a loading control. Bottom: STAT and phospho-STAT (pSTAT) levels were also assessed following stimulation with 1,000 U/ml of either IFN–α2b or –γ for 0.5 h on F-SV40 cells from two healthy controls (C1 and C2), the IRF9-deficient patient (IRF9−/−), her mother (IRF9+/−), a STAT1-deficient patient (STAT1−/−), a STAT2-deficient patient (STAT2−/−), an IFNGR2-deficient patient (IFNGR2−/−), and an IRF7-deficient patient (IRF7−/−). Representative results of five independent experiments are shown. (C) WB of IRF9 in IRF9-deficient U2A cells stably transfected with indicated variants (green: variants reported to be loss-of-function in in vitro assays, blue: variants found in-house, red: patient). GAPDH was used as loading control. Representative results of four independent experiments are shown. (D) WB of IRF9 in patient F-SV40 cells stably transfected with indicated variants. GAPDH was used as loading control. Representative results of four independent experiments are shown. (E) WB analysis of IRF9 localization in F-SV40 cells from two healthy controls (C1 and C2), the IRF9-deficient patient (IRF9−/−), her mother (IRF9+/−), a STAT1-deficient patient (STAT1−/−), a STAT2-deficient patient (STAT2−/−), an IFNGR2-deficient patient (IFNGR2−/−), and an IRF7-deficient patient (IRF7−/−). GAPDH and LaminA/C were used as loading controls. Representative results of three independent experiments are shown. (F) Reporter assays of ISRE or GAS-dependent firefly luciferase tested in U2A cells stimulated with 1,000 U/ml of either IFN-α2b or -γ for 16 h after being stably transfected with indicated variants (green: variants reported to be loss-of-function in in vitro assays, blue: variants found in-house, red: patient). The specific response to IFN stimulation was calculated by the ratio of firefly luciferase reporter gene activity to constitutively expressed renilla luciferase activity (RLU, relative luciferase ratio). Representative results of three independent experiments are shown. (G) EMSA analysis of ISRE and GAS binding by IFN-stimulated B-LCLs from three healthy controls (C1, C2, and C3), the IRF9-deficient patient (IRF9−/−), her mother (IRF9+/−), a STAT1-deficient patient (STAT1−/−), a STAT2-deficient patient (STAT2−/−), and an IRF7-deficient patient (IRF7−/−). Representative results of three independent experiments are shown.