Figure 5.

PLpro-TM reduces the ubiquitination levels of signaling molecules. The plasmids expressing Flag-tagged RIG-I (A), TRAF3 (B), STING (C), TBK1 (D), and Myc-tagged IRF3 (E) together with HA-Ub-K63 were co-transfected into HEK293T cells in the presence and in the absence of V5-tagged PLpro-TM. Cells were incubated for 24 h after transfection and treated with 25 μmol/L MG132 for 4 h prior to the harvest of the cell lysate. The cell lysate was immunoprecipitated with the indicated antibodies. The products were immunoblotted using anti-HA to evaluate K63-linked ubiquitinated proteins (upper panels). The whole cell lysate (WCL) was blotted to evaluate the expression of PLpro-TM (bottom panels). Each experiment was repeated at least 3 times. The asterisk indicates the nonspecific band from cellular proteins with high affinity with Agarose