Fig 4.
K219 and K70 are the primary and secondary target sites in NS1, respectively, for both SUMO1 and SUMO2/3 SUMOylation. HEK293A cells were cotransfected with (+) or without (−) the indicated combinations of mammalian expression plasmids, including expression plasmids for T7T7-tagged NS1 (T7T7NS1), the single and double lysine mutant forms of NS1 (T7T7NS1K70A, T7T7NS1K219A, and T7T7NS1K70AK219A), an expression construct for the human de-SUMOylating enzyme SENP1, an empty expression vector used to equalize the total amount of DNA used to transfect the cells, and the dicistronic expression constructs Dual S1/I/Ubc9 and Dual S1Q94P/I/Ubc9 (S1Q94P is a mutant form of SUMO1 that prevents its de-SUMOylation upon its conjugation to a target) (A to C) or the dicistronic expression constructs Dual S3/I/Ubc9 and Dual S3Q89P/I/Ubc9 (S3Q89P is a mutant form of SUMO3 that prevents its de-SUMOylation upon its conjugation to a target) (D to F). Cell extracts were collected and analyzed by SDS-PAGE and immunoblotting using either anti-T7 MAb (A and D), anti-SUMO1 MAb (B), anti-SUMO2 PAb (E), or anti-GAPDH MAb (C and F). NS1His-S-SUMO, NS1 SUMOylated with His- and S-tagged SUMO; NS1SUMO, NS1 SUMOylated with endogenous SUMO; HMW, high molecular weight.