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. 2021 Jul 23;13(8):1439. doi: 10.3390/v13081439

Figure 5.

Figure 5

TRIM25 E3 ubiquitin ligase activity was impaired by the presence of SARS-CoV-2 N protein. The transient expression of SARS-CoV-2 N protein affected the ability of TRIM25 to mediate RIG-I ubiquitination in Lenti-X 293T cells. (A) Cells transfected with plasmids expressing FLAG-RIG-I, HA-TRIM25, HA-K63-only ubiquitin mutant and, where indicated, the SARS-CoV-2 N protein, were treated with the proteasome inhibitor MG-132 for 12 h. Lysates, prepared under denaturing conditions as described in the Material and Methods section, were immunoprecipitated with anti-FLAG magnetic beads, and precipitated proteins were resolved by SDS-PAGE. Pulled proteins were detected by immunoblotting with anti-HA or anti-FLAG monoclonal antibodies. Blots are representative of at least two biologically independent experiments. (B) The RIG-I ubiquitination rate was determined by densitometric analysis calculating the ratio between mono- and poly-ubiquitinated RIG-I (anti-HA) and relative RIG-I levels (anti-FLAG). Fold changes in RIG-I ubiquitination were calculated for N-containing samples relatively to the control sample. Results are expressed as mean (n = 3) fold change ± standard deviation (SD), and significance was determined by p-value (*** p < 0.005).