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. 2020 Mar 4;217(5):e20191174. doi: 10.1084/jem.20191174

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© 2020 Cai et al.

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Figure 8. — USP22 deubiquitinates and stabilizes KPNA2. (A) Immunoprecipitation (IP, with rabbit anti-KPNA2) and immunoblot (with mouse anti-USP22, anti-IRF3, and rabbit anti-KPNA2 or anti-pIRF3, goat anti-rabbit, and anti-mouse IgG[H+L] antibody) of THP-1 cells that were left uninfected or infected with SeV or HSV-1 for 4–8 h. Cell lysates were analyzed by immunoblot with antibodies against the indicated proteins. (B) Denature-IP (with anti-KPNA2) and immunoblot analysis (with anti-Ub, anti-KPNA2, anti-USP22 or anti-GAPDH) of Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl MEFs treated with 4-OHT (1 µM) for 3 d and reconstituted with empty vector (phage), USP22, or USP22(C185S) followed by infection with SeV or HSV-1 for 0–8 h. (C) Immunoblot analysis of KPNA2, USP22, GAPDH, and Lamin B1 in the cytoplasmic (30 µg) and nuclear (45 µg) extracts of Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl MEFs treated as in B followed by infection with VSV for 0–8 h. (D) Immunoblot analysis of KPNA2, FLAG-USP22, and GAPDH in Cre-ER Usp22^fl/fl MEFs treated as in B followed by infection with VSV for 0–12 h. (E) Immunoblot analysis of KPNA2, USP22, and GAPDH in Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl MEFs treated as in B followed by infection with VSV for 12 h in the presence of MG132 or BafA1 for 11 h. (F and G) Immunoblot analysis of KPNA2, USP22, ATG7, and GAPDH of Cre-ER Usp22^fl/+ and Cre-ER Usp22^fl/fl MEFs treated with 4-OHT for 3 d and transfected with siATG7 (F) or siNDP52 (G) for 36 h followed by infection with VSV for 8 h. Data are representative of two independent experiments.