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. 2013 Oct 30;288(51):36312–36327. doi: 10.1074/jbc.M113.486845

FIGURE 6.

FIGURE 6.

Regulation of UBC9- and SUMO1-dependent repression by SUMO non-covalent interactions. A, schematic representation of the firefly Luciferase reporter plasmid and Gal4-effector constructs used in the repression assay. The Luciferase reporter is under the control of the strong SV40 promoter and SV40 enhancer sequence and contains five consensus Gal4 UAS (5xGal4UAS). Effectors proteins UBC9 and SUMO1 and mutants fused to Gal4DBD are shown. B, transcriptional activity of Gal4DBD and Gal4DBD-UBC9 proteins directly recruited on the Luciferase reporter plasmid. Increasing amounts (100, 200, and 400 ng) of the effector constructs were used. C, transcriptional activity of Gal4DBD and Gal4DBD fusion proteins (UBC9, UBC9-C93S, UBC9-K14R, UBC9-P69A, UBC9-R17A, and UBC9-H20D) directly recruited on the Luciferase reporter plasmid. Increasing amounts (200 and 400 ng) of the effector constructs were used. The expression of Gal4DBD-UBC9 and Gal4DBD-UBC9 mutants (200 ng) was monitored by immunoblotting with an anti-Gal4DBD antibody. D, transcriptional activity of Gal4DBD and Gal4DBD fusion proteins (UBC9, UBC9-D100A-K101A, UBC9-P128A, and UBC9-Y134A) directly recruited on the Luciferase reporter plasmid. Increasing amounts (100, 200, and 400 ng) of the effector constructs were used. The expression of Gal4DBD-UBC9 and Gal4DBD-UBC9 mutants (200 ng) was monitored by immunoblotting with an anti-Gal4DBD antibody. In B–D, the -fold repression is the ratio of Luciferase activity measured in the presence of Gal4DBD control divided by the activity measured in the presence of the Gal4DBD-UBC9 fusions constructs. In each case, the -fold repression was normalized to account for transfection efficiency. Error bars represent the S.D. for at least three independent experiments performed with duplicate or triplicate samples. IB, immunoblot. E, transcriptional activity of Gal4DBD fused to the non-conjugate-able form of SUMO1 and SUMO1 mutants (SUMO1-F36A and SUMO1-E67R) directly recruited on the Luciferase reporter plasmid. 200 ng of the effector constructs were used. The percentage change in -fold repression of the Gal4DBD-SUMO1 constructs is expressed relative to the Luciferase activity of the Gal4DBD control fixed to 1-fold repression; thus, the activity of Gal4DBD control corresponds to 0% change in -fold repression. Error bars represent S.D. for at least two independent experiments performed with duplicate samples. The expression of Gal4DBD-SUMO1 constructs was monitored by immunoblotting with an anti-Gal4DBD antibody.