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. 2022 Oct 2;23(19):11692. doi: 10.3390/ijms231911692

Figure 4.

Figure 4

Suppression of the MDA5-mediated IFN-β induction by SARS-CoV-2 and IBV nsp3. (a) Western blot analysis of the expression of Flag-MDA5 co-transfected with Hibit-tagged SARS-CoV-2 or IBV nsp3. HEK293T cells were transfected either with Flag-MDA5 alone or together with Hibit-tagged SARS-CoV-2 or IBV nsp3. Cells were harvested at 24 h post-transfection and subjected to Western blot analysis with anti-FLAG and anti-Hibit antibodies, respectively. Beta-actin was included as the loading control. Sizes of protein ladders in kDa are indicated on the left. (b) Suppression of the MDA5-mediated IFN-β induction by SARS-CoV-2 and IBV nsp3. Total RNA was extracted from cells harvested in (a), and the IFN-β levels were determined by RT-qPCR after normalization with the GAPDH mRNA extracted from cells transfected with the empty vector. Significance levels were presented by the p-value (ns, non-significance, **, p < 0.01; ***, p < 0.001). (c) Western blot analysis of the expression of Flag-MDA5 co-transfected with Hibit-tagged full-length or truncated SARS-CoV-2 nsp3. Cells were harvested at 24 h post-transfection and subjected to Western blot analysis with anti-FLAG and anti-Hibit antibodies, respectively. Beta-actin was included as the loading control. Sizes of protein ladders in kDa are indicated on the left. (d) Effects of co-expression of MDA5 and the full-length or truncated SARS-CoV-2 nsp3 on the MDA5-mediated IFN-β induction. Total RNA was extracted from cells harvested in (c), and the IFN-β levels were determined by RT-qPCR after normalization with the GAPDH mRNA extracted from cells transfected with the empty vector. Significance levels were presented by the p-value (ns, non-significance, ***, p < 0.001).