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. 2021 Aug 27;118(37):e2107108118. doi: 10.1073/pnas.2107108118

Fig. 2.

Fig. 2.

The 3Clpro cleaves RNF20 at residue Q521 via its protease activity. (A) Cleavage of ectopic RNF20 by 3Clpro. Vector expressing RNF20 (without Tag) was cotransfected with control vector or HA-3Clpro into HEK293T cells for 24 h. Cleavage of RNF20 was determined by Western blot using the specific antibody against the N terminus of RNF20. (B) Immunoblotting detection of endogenous RNF20 cleavage. HEK293 cells were transfected with control vector or HA-3Clpro for 24 h. Cell lysates were harvested for analyzing cleavage of endogenous RNF20 with the antibody against the N terminus of RNF20. (C) Endogenous RNF20 cleavage upon SARS-CoV-2. Vero E6 cells were infected with SARS-CoV-2 at MOI 2 for 24 and 48 h. Cell lysates were analyzed by Western blot to show the cleavage of endogenous RNF20. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. (D and E) The positive control vector (carrying SAVLQSGFRK, also used in Fig. 1E) or RNF20 (without Tag) were transfected into HEK293T with HA-3Clpro for 20 h and then treated with either dimethyl sulfoxide (DMSO) or main protease inhibitor GC-376 (50 µm) for 12 h prior to measuring the cleavage of the indicated protein. (F) HEK293T cells were transfected with HA-tagged 3Clpro for 20 h, and then treated with DMSO, MG132 (10 µm), MLN4924 (1 µm) for 6 h or GC-376 (50 µm) for 12 h, followed by immunoblotting analysis with the indicated antibodies. (G) GFP7/8 and GFP-BIRC6-103 were cotransfected with wild-type 3Clpro or enzymatic dead mutant (C145A) 3Clpro into HEK293T cells for 24 h. Western blotting was performed to analyze the cleavage of the indicated protein. (H) HEK293T cells were transfected with wild-type 3Clpro or mutant C145A for 24 h and then subjected to immunoblotting analysis of endogenous RNF20 cleavage with the indicated antibodies. (I) HEK293T cells were cotransfected with a plasmid expressing FLAG-RNF20 or its mutant (Q521A) alone or in combination with a plasmid expressing HA-3Clpro for 24 h, and the cell lysates were analyzed by Western blotting. (J) Analysis of the protein sequences across species for RNF20 cleavage sites using Clustal Omega online service.