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. 2021 Apr 28;17(4):e1009530. doi: 10.1371/journal.ppat.1009530

Fig 7. DDX5 negatively regulated DHX58-mediated TBK1 and p65 pathways in innate immunity.

Fig 7

(A, B): Immunoblot of DHX58-mediated TBK1 and p65 pathways in DDX5-knockdown MEFs (A) or DDX5-expressing MEFs (B). MEFs were transfected with DDX5 siRNA or METTL3 siRNA (siNC) for 48 h (A), or with DDX5 or METTL3 expression (control vector) for 24 h (B); then, cells were infected with VSV for 0, 4, and 6h, lysed, and collected to detect DDX5, METTL3, p-IKKγ, IKKγ,p-p65,p65,p-TBK1, TBK1 and p-IRF7 via western blot. (C, D): Nuclear transfer of IRF7 was observed by IFA and CLSM. MEFs were transfected with DDX5-expression plasmids (control vector) for 24 h (C) or with DDX5 siRNA (siNC) for 48 h (D), infected with VSV for 8 h, and then subjected to IFA with anti-IRF7 monoclonal antibody followed by Alexa Fluor 488 F(ab′)2 fragment of goat anti-mouse IgG (H+L). Nuclei were stained with DAPI. Normal MEFs (WT) were used as the control. Slices were observed by LSCM. Scale bars, 20 μm. (E, F): Nuclear transfer of p65 observed by IFA and CLSM. MEFs were transfected with DDX5-expression plasmids (E) or with DDX5 siRNA (F), infected with VSV for 8h, and subjected to IFA with anti-p65 monoclonal antibody followed by Alexa Fluor 488 F (ab′)2 fragment of goat anti-mouse IgG (H+L). Nuclei were stained with DAPI. Normal MEFs (WT) were used as the control. Slices were observed by LSCM. Scale bars, 20 μm.