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. 2016 Apr 14;90(9):4308–4319. doi: 10.1128/JVI.00223-16

FIG 2.

FIG 2

DENV NS5 protein is a target for SUMO modification. (A) HEK293T cells were cotransfected with Ubc9 and each HA-tagged DENV viral protein, with or without EGFP-tagged SUMO1, for 48 h. The cell lysates were subjected to immunoprecipitation (IP) by anti-HA agarose and WB analysis with anti-HA antibody. (B) The in vitro SUMOylation assay was reconstituted with recombinant SUMO E1 (Aos1/Uba2), SUMO E2 (Ubc9), His-tagged SUMO1, and immunopurified NS5 in the presence (+) or absence (−) of ATP as described in Materials and Methods. The reaction mixture was analyzed by WB with anti-NS5 antibody. (C) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 or left untransfected, as indicated, for 48 h. Transfectants were collected and subjected to analysis by IP-WB with the indicated antibodies. (D) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 together with SENP1 or SENP2 for 48 h as indicated. Cell lysates were harvested for IP-WB analysis with anti-HA antibody. Arrow, SUMOylated NS5 protein. (E) N18 cells were infected with DENV (MOI of 10) for 6 h and then transfected with EGFP-tagged SUMO1 or HA-tagged GFP. After 18 h of transfection, cells were lysed and analyzed by IP-WB with anti-GFP and anti-NS5 antibodies. (F) HEK293T cells were cotransfected with NS5 and Ubc9 plus Flag-tagged SUMO1 or SUMO2 for 48 h. Transfectants were harvested and subjected to analysis by IP-WB with the indicated antibodies. IB, immunoblot.