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. 2021 Apr 26;95(10):e02194-20. doi: 10.1128/JVI.02194-20

FIG 6.

FIG 6

KSHV ORF61 and ORF59 are potential FAT10ylation substrates. (A) 293T cells were cotransfected with plasmids expressing His-FAT10 (20 kDa) and either 2xStag-ORF59-3xFLAG (Stag-ORF59; 42 kDa) or 2xStag-ORF61-3xFLAG (Stag-ORF61; 88 kDa). At 48 h posttransfection, the cells were lysed in RIPA buffer and subjected to pulldown assays (PD) using S-protein beads. The FAT10ylation of ORF61 and ORF59 was confirmed by immunoblotting (IB) using anti-His antibody. The FAT10ylated ORF59 (approximately 60 kDa) and ORF61 (approximately 110 kDa) are indicated by an asterisk. TCL, total cell lysate. (B) Effect of UBE1L2 knockout on the FAT10ylation of ORF61. 293T cells were transfected with the UBE1L2 CRISPR/Cas9 KO plasmid (UBE1L2-KO) or control CRISPR/Cas9 plasmid (control) and cultured for 4 days. KO plasmid-transfected cells were cotransfected with His-FAT10 and 2xStag-ORF61-3xFLAG plasmids. Cells were subjected to pulldown assay at 48 h posttransfection. The FAT10ylated ORF61 are both indicated by an asterisk. (C) 293T cells were cotransfected with plasmids expressing His-FAT10 and either 2xStag-ORF37-3xFLAG (Stag-ORF37; 55 kDa) or 2xStag-ORF56-3xFLAG (Stag-ORF56; 96 kDa).