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. 2022 Jun 23;13:914971. doi: 10.3389/fmicb.2022.914971

Figure 3.

Figure 3

The 87Q-88G is the only site in TRAF3IP3 that is cleaved by 3Cpro. (A) Schematic representation of the potential 3Cpro cleavage sites in TRAF3IP3. (B) HEK293T cells were transfected with HA-TRAF3IP3 1–301 or 302–551 and Myc-3C. Forty-eight hours later, Western blotting was used to detect the proteins. (C) HA-TRAF3IP3 wild-type or G88A mutant was co-transfected with Myc-3C or mutant C147S into HEK293T cells. Cells were harvested 48 h after transfection for Western blotting analysis. (D) HEK293T cells were transfected with HA-TRAF3IP3 wild-type or G88A mutant. Cells were harvested 48 h after transfection and lysates were incubated with purified 3Cpro or mutant 3C-C147S for 90 min at 30°C. Loading buffer was added to stop the reaction, followed by Western blotting detection of proteins. (E) HA-TRAF3IP3 single-mutant S303A or double-mutant G88A S303A (M2) were co-transfected with Myc-3C or mutant C147S into HEK293T cells. Cells were harvested 48 h after transfection for Western blotting. (F) HA-TRAF3IP3-S303A or M2 was transfected into HEK293T cells. The rest of the experiment was the same as the in vitro cleavage in (D).