Fig. 6
From: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing
Impaired TBK1 activation after RLR or TLR3 stimulation in OPTN-deficient primary cells. a Primary MEFs isolated from WT or OPTN470T mice were infected with Sendai virus (SeV) for the indicated times. Cell lysates were then analyzed by immunoblotting with antibodies against the indicated proteins. * Indicates a non-specific band. b, c Primary MEFs isolated from WT or OPTN470T mice were infected with SeV for the indicated times. IFNB1 and IL-6 mRNA levels were assessed by RT-qPCR with normalization against GAPDH. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT MEFs in Student’s t test). ns, not significant; AU, arbitrary unit. d, e Primary MEFs isolated from WT or OPTN470T mice were either left unstimulated or infected with SeV for 6 or 8 h. The production of IFNβ and IL-6 was then analyzed by ELISA in the cell supernatant. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT MEFs in Student’s t test). ns, not significant. f Primary MEFs isolated from WT or OPTN470T mice were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was assessed by immunofluorescence staining and aggregate counting. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). **0.001 < P < 0.01 versus WT MEFs (Student’s t test). g BMDM isolated from WT or OPTN470T mice were stimulated with poly(I:C) (1 μg/mL) for the indicated times. Cell lysates were then analyzed by immunoblotting with antibodies against the indicated proteins. h–k BMDM isolated from WT or OPTN470T mice were stimulated with poly(I:C) (1 μg/mL) for the indicated times. IFNB1 and IL-6 mRNA levels were then assessed by RT-qPCR with normalization against GAPDH (h, i), or the production of IFNβ and IL-6 was analyzed by ELISA in the cell supernatant (j, k). The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT BMDM in Student’s t test). ns, not significant; AU, arbitrary unit