FIG. 6.

WNVCp contains the region from amino acid 90 to 105 responsible for MKRN1-dependent degradation. (A) Requirement of the N terminus of WNVCp for MKRN1 interaction. A plasmid expressing FLAG-MKRN1 was transfected with or without HA-WNVCp or its deletion mutants in 293T cells. Cell lysates were immunoprecipitated with anti-FLAG antibodies. Whole-cell lysates (WCL) and immunoprecipitates (IP) were detected with anti-HA and anti-FLAG antibodies. The asterisk indicates heavy chains of antibodies. WT, wild type. (B) Requirement of the 90-105 region of WNVCp for MKRN1-dependent degradation. Mock vector or plasmids expressing FLAG-MKRN1, HA-WNVCp, or HA-WNVCp deletion mutants were transfected into H1299 cells. pEGFP-C2 expressing enhanced GFP was cotransfected as a transfection control. The lysates were detected with anti-HA antibodies, anti-FLAG antibodies, and anti-GFP antibodies. The protein quantification was performed as described for Fig. 1. Error bars indicate standard deviations. (C) Alignment of WNVCp orthologs containing conserved lysine residues. Gray shading indicates lysine residues conserved throughout flaviviruses.