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. 2020 May 26;30(10):914–927. doi: 10.1038/s41422-020-0341-6

Fig. 2. Knockdown of USP29 impairs HSV-1-induced innate immune signaling.

Fig. 2

a Immunoblot analysis (with anti-FLAG or anti-HA) of HEK293 cells transfected for 36 h with plasmids encoding FLAG-tagged USP29 and HA-β-Actin and either USP29-targeting shRNA (shUSP29#1 and shUSP29#2) or control shRNA (Con) to test the knockdown efficiency of shRNA (upper two panels). Immunoblot analysis (with anti-USP29 or anti-β-Actin) of THP-1 cells stably transfected with plasmids encoding control (Con) shUSP29 (#1 or #2) (lower two panels). b qRT-PCR analysis of IFNB, TNF, IL6, and IP10 in THP-1 cells stably transfected with control shRNA, shUSP29#1 or shUSP29#2 that were infected with HSV-1 for 0–8 h. c qRT-PCR analysis of IFNB, IP10, and TNF in THP-1 cells stably transfected with control shRNA, shUSP29#2 that were mock transfected or transfected with DNA90, HSV60 or HSV120 for 6 h. d qRT-PCR analysis of IFNB, TNF and IP10 mRNA in THP-1 cells stably transfected with control shRNA or shUSP29#2 that were left untreated (Mock) or treated with cGAMP for 4 h. e ELISA analysis of IFN-α, IFN-β, IL-6 and TNF in the supernatants of THP-1 cells stably transfected with control shRNA or shUSP29#2 that were infected with HSV-1 for 0–24 h, or mock transfected or transfected with DNA90, HSV60 or HSV120 for 12 h. f Immunoblot analysis of phosphorylated and total TBK1, IRF3 and IκBα, and USP29 and GAPDH in THP-1 cells stably transfected with control shRNA or shUSP29#2 that were infected with HSV-1 for 0–6 h. *P < 0.05; **P < 0.001; ***P < 0.001 (analysis of two-way ANOVA followed by Bonferroni post-test or two-tailed Student’s t-test). Data are representative of three independent experiments (means ± SD in b–e).