IFNλ4p179 and IFNλ3 have similar biological activities. (A) Schematic of the gene structure of protein-coding splice variants of IFNL4 generated by alternative splicing. (B) Immunoblot of HA-tagged IFNλ3 and IFNλ4 isoforms overexpressed in Huh7 cells. (C) Immunoblot of PNGase F-treated lysates from cells overexpressing HA-IFNλ4 isoforms or HA-IFNλ3. (D) Immunoblot of cell lysates, neat supernatants, and supernatants concentrated by trichloroacetic acid (TCA) treatment. (E) Immunoblot of TCA-treated supernatants of Huh7 cells overexpressing IFNλ4 isoforms. (F) Luciferase reporter assay measuring ISRE activity in wild-type and IFNLR1^−/− Huh7 cells overexpressing IFNλ3 or IFNλ4 isoforms. (G) Luciferase reporter assay measuring ISRE activity in Huh7 cells treated with IL-10R2–neutralizing antibody and overexpressing IFNλ4p179 and IFNλ3. (H) MX1 expression in wild-type and IFNLR1^−/− Huh7 cells overexpressing IFNλ4p179 and IFNλ3. (I–K) MX1 expression in Huh7 cells overexpressing IFNλ4 isoforms and treated with either IFNλ3 (I and J) or IFNβ (K). (J) MX1 expression after co-transfection of a GFP plasmid along with the IFNL4 and IFNL3 overexpression constructs sorting of GFP⁺ cells directly before IFNλ3 stimulation for 9 h. (L) Renilla luciferase reporter activity measured in Huh7 cells overexpressing IFNλ3 or IFNλ4 isoforms infected with HCV tagged with Renilla luciferase. Experiments are representative of at least two to three biological replicates. (B–E) Filled arrowheads, glycosylated forms; empty arrowheads, deglycosylated forms. Statistical analysis was performed using one-way ANOVA with multiple comparisons against EV-transfected cells (L). ***, P < 0.001; ns, not significant.