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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: J Immunol. 2017 Oct 25;199(11):3808–3820. doi: 10.4049/jimmunol.1700807

Fig. 2. IFN-λ4 activity requires secretion but at very low levels.

Fig. 2

(A) Representative flow cytometry plots for expression of IFN-λ3-GFP and IFN-λ4-GFP induced with doxycycline (Dox) for 24 hrs in corresponding stable HepG2 cell lines; expression of IFN-λ4-GFP was readily detectable, while IFN-λ3-GFP was detectable only after treatment with GolgiStop for 4 hrs to block protein secretion. (B) Kinetics of ISRE-Luc activation in stable HepG2 cells expressing IFN-λ4-GFP or IFN-λ3-GFP co-cultured in a 1:1 ratio with HepG2-ISRE-Luc cells. (C) Concentration of GFP measured by ELISA (n=2) in culture media of the experiment described in (B); shown one of two independent experiments. (D) Western blotting for GFP in culture media of stable inducible HepG2 cells expressing IFN-λ4-GFP (not detectable) or IFN-λ3-GFP (detectable, marked by arrow) from experiment described in (B). M - protein size marker. (E) The effect of secreted IFN-λ4 is tested in stable HepG2 cells expressing IFN-λ4-GFP co-cultured in a 1:1 ratio with HepG2-ISRE-Luc cells. The effect of both secreted and intracellular IFN-λ4 is tested in stable HepG2 cells expressing IFN-λ4-GFP and ISRE-Luc reporter. Cells were induced with doxycycline (Dox) for 24 hrs to express IFN-λ4-GFP in the presence of 20 μg/ml of rabbit antibodies - blocking anti-IFN-λ4 or IgG control, shown one of two independent experiments.