Fig. 2

Slug is covalently modified by SUMO-1. a Identification of Slug SUMOylation using the in vitro SUMOylation assay system. The purified proteins were incubated and then analyzed by immunoblotting using the indicated antibodies. b Exogenous Slug SUMOylation. The lysates of HEK293T cells transfected with the indicated plasmids and treated or not treated with NEM were collected and subjected to immunoprecipitation using an anti-Flag antibody. The results were analyzed by immunoblotting with the indicated antibodies. c, d Endogenous Slug SUMOylation. The lysates of CL1–5 cells treated with NEM were immunoprecipitated with an anti-Slug (c) or anti-SUMO-1 antibody (d); the precipitates were individually resolved in reducing (c) or non-reducing (d) sample buffer. Immunoblotting was performed using anti-Slug and anti-SUMO-1 antibodies to examine the endogenous SUMOylation of Slug. e-g Lysates of HEK293T cells cotransfected with different fragments or mutant forms of Slug were harvested and then subjected to immunoblotting with an anti-Flag antibody. The results were analyzed by immunoblotting with the indicated antibodies. The asterisk and arrowhead indicate Slug modified and not modified with SUMO-1, respectively