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. 2020 Jan 24;12(2):137. doi: 10.3390/v12020137

Figure 2.

Figure 2

The effect of SV40 early region on exogenous and endogenous cGAS and STING expression. (A) The 293T cells were co-transfected with 100 ng of hemagglutinin (HA) epitope-tagged human STING (pUNO1-hSTING-HA3x) with increasing amounts of SV40 early region (ER) plasmid (100 ng, 500 ng, and 1 µg). The anti-HA antibody was used for the detection of SV40 ER and STING. Actin or Tubulin was probed as a loading control as indicated. (B) The 293T cells were co-transfected with 100 ng of HA epitope-tagged human cGAS (pUNO1-hcGAS-HA3x) with increasing amounts of SV40 ER plasmid (100 ng, 500 ng, and 1 µg). (C,D) 293 cells were transfected with the same increasing amounts of SV40 ER plasmid (100 ng, 500 ng, and 1 µg). Western blot analysis for endogenous levels of (C) cGAS and STING, and (D) TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) was assessed. (E) The 293 cells were transfected with 500 ng of SV40 ER, SV40 large T antigen (LT), or SV40 LT AxAxA (mutations in the LxCxE motif). The endogenous expression of STING was assessed by western blot analysis. (F) The 293 cells were co-transfected with 1 μg of empty vector or untagged SV40 ER. The figure shows western blot analyses for SV40 LT with a monoclonal SV40 T-antigen antibody, endogenous STING, and Actin (loading control). (G) The 293 cells were transfected with 1 µg of SV40 ST, 17kT, or LT plasmid. Western blot analysis was performed at 24 hours post-transfection for endogenous STING expression, HA epitope-tagged SV40 ST, 17kT or LT, and Tubulin (loading control). All western blots shown in this Figure are representative of at least three independent experiments.