Additional file 2: of Hypoxia-induced Slug SUMOylation enhances lung cancer metastasis

Figure S2. The activities of Slug mutants. (a) Mutation of individual lysine affects the SUMOylated level of Slug. HEK293T cells were cotransfected with plasmids encoding different 3xFlag-tagged Slug mutants and GFP-tagged SUMO-1. The lysates were used to examine the SUMOylation levels by immunoblotting with anti-Flag antibodies. (b) Different levels of SUMOylation between Slug mutants. HEK293T cells were transfected with expression vectors encoding GFP-tagged SUMO-1 and different 3xFlag-tagged Slug mutants (22 M, all lysines were replaced with arginines; 5 M: lysines at 239, 240, 244, 248, and 258 were replaced with arginines; 6 M: lysines at 188, 239, 240, 244, 248, and 258 were replaced with arginines). These lysates were also examined by immunoblotting with anti-Flag antibodies. The asterisk and arrowhead indicate Slug modified and not modified by SUMO-1, respectively. (c) The transcriptional repression activity of wild-type and mutant Slug proteins. HEK293T cells were cotransfected with the SBS–Gal4–luciferase reporter and Gal4–VP16 activator expression plasmids together with the wild-type or mutant Slug expression plasmid (8 M: lysines at 135, 145, 188, 239, 240, 244, 248, and 258 were replaced with arginines), and the luciferase assay was performed to determine the transcriptional repression activity of Slug. Immunoblotting results are presented alongside the luciferase assay results to demonstrate the expression of the Slug mutant proteins. (d) The DNA-binding activity of wild-type and mutant Slug proteins. The wild-type and mutant Slug proteins used in the EMSA were produced using an in vitro transcription/translation system. The protein expression levels were evaluated by immunoblotting with anti-Slug antibodies (top panel). Phosphor image analysis of the EMSA gel showing 32P-labeled E-box oligonucleotides incubated with in vitro-translated proteins (4 μl) or with Slug antibodies (Ab: antibody, 0.3 μg) (bottom panel). (PDF 152 kb)