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. Author manuscript; available in PMC: 2018 May 24.
Published in final edited form as: Biochem J. 2017 Oct 10;474(20):3543–3557. doi: 10.1042/BCJ20170548

Figure 7. Ubiquitination is necessary for efficient IFNGR1 signaling.

Figure 7

(a) Several HEK cell lines with IFNGR1 knocked out by CRISPR–Cas9 (HEK Cas9) were transfected with IFNGR1 to rescue IFNGR1 signaling. Cells were transfected with empty vector or IFNGR1 for 48 h and treated with 50 ng/ml IFN-γ 15 min prior to harvest. Cells were then harvested and probed for IFNGR1, and pSTAT1. HEK Cas9 IFNGR1 KO cell line 1#1 was used in subsequent experiments and cells were treated as described above. (b) HEK Cas9 cells were transfected with various IFNGR1 lysine to arginine mutant plasmids and probed for IFNGR1 levels and phosphorylated STAT1 (pSTAT1). (c) HEK cells were transfected with indicated amounts of Wt IFNGR1 or the K-R (277, 279, 285R) mutant plasmids for 48 h, harvested and probed for IFNGR1 and pSTAT1 by immunoblotting. (d) Densitometry of pSTAT1 in HEK transfected with 0.02, 0.1, 0.20, 0.5 μg/ml of Wt or K-R mutant IFNGR1. *P < 0.05 Wt vs. K-R by Student’s t-test. Below: pSTAT1 signaling normalized to total protein expression at 0.10, 0.20, 0.50 μg/ml DNA transfection. *P < 0.05 Wt vs. K-R by Student’s t-test. (e) HEK cells were transfected with 0.5 μg/ml plasmids encoding V5-tagged IFNGR1 Wt or K-R, for 48 h. Cells were then treated with membrane impermeable biotin, harvested, and probed for IFNGR1 to determine plasma membrane localization. Wt (No Bio) = transfected, no biotin control, UT, untransfected. Below: pSTAT1 normalized to plasma membrane IFNGR1 expression in cells transfected with Wt or K-R mutant IFNGR1. (f) IFN-γ stimulated gene induction in HEK cells. HEK cells and HEK IFNGR1 Cas9 KO cells were seeded into 6-well plates. HEK IFNGR1 Cas9 KO cells were transfected with empty vector, Wt IFNGR1 0.5 μg/ml, for 24–48 h. Cells were then treated with IFN-γ (50 ng/ml) for indicated times, harvested for RNA and cDNA, and probed for IFN-γ stimulated gene induction by qRT-PCR. (g) HEK IFNGR1 Cas9 KO cells were transfected with Wt IFNGR1 or K-R mutant as described above. n = 3–4 independent experiments ± SE. *P < 0.05 K-R vs. Wt by ANOVA.