Figure 6.

Knockout of USP21 expression enhances antiviral response in MEFs. (A) WT and USP21^−/− MEFs were transfected with IFN-β or ISRE reporter along with or without RIG-I-CARD for 24 h, and then left uninfected or infected with SeV or transfected with poly (I:C) for 12 h before luciferase activity was measured. (B) MEFs were treated with SeV for the indicated time points. The mRNA levels of IFN-α/β were measured by real-time RT-PCR. (C) MEFs were infected with VSV-eGFP (moi = 0.1) for 48 h and GFP⁺ cells were imaged. Bar, 50 µm. (D) MEFs were infected with VSV for the indicated MOI for 48 h and relative cell viability was measured by CCK8 kit. (E) MEFs were infected with VSV or SeV for the time points indicated. Cell lysates were immunoblotted by anti–p-IRF3, anti-IRF3, anti-USP21, anti–p-IKKα/β, anti-IKKβ, and anti–β-actin antibodies. (F) MEFs were infected with SeV for the indicated time points. Cell lysates were immunoblotted by anti-SeV antibodies. (G) MEFs were infected with SeV for the indicated time points. Endogenous USP21 were immunoprecipitated with anti-USP21 antibodies and immunoblotted with anti–RIG-I antibodies. (H) MEFs were infected with SeV for the indicated time points. RIG-I proteins in the cell lysates were immunoprecipitated with anti–RIG-I antibodies and immunoblotted with anti-Ub antibodies. Error bars indicate ±SD in duplicate experiments. Data are representative of two (B–D and F) or three (A, E, G, and H) independent experiments.