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. 2001 Dec 1;15(23):3088–3103. doi: 10.1101/gad.944801

Figure 5.

Figure 5

PIASy represses LEF1 activity. (A) PIASy represses LEF1 activity from a multimerized LEF1 reporter. 293T cells were transfected with 1 μg of a LEF1 luciferase reporter construct containing multimerized LEF1-binding sites together with expression constructs encoding for β-galactosidase (25 ng; for normalization), LEF1 (30 ng), β-catenin (1 μg), or increasing amounts of T7-PIASy (0.1, 0.3, or 1 μg), as indicated. For this and subsequent experiments, the levels of luciferase or CAT activity were normalized for β-galactosidase activity and are expressed as fold activation relative to the level of luciferase or CAT activity from cells transfected with the reporter construct alone. All of the transfection experiments were performed at least three times, and the results of representative experiments are shown. (B) PIASy represses LEF1-dependent Twn reporter activity. NMuMG epithelial cells were transfected with 2 μg of the Twn luciferase reporter construct containing either wild-type (Twn-Luc) or mutated (ΔLEF-Twn-Luc) LEF1-binding sites together with expression constructs encoding for β-galactosidase (0.5 μg; for normalization), LEF1 (0.5 μg), β-catenin (5 μg), or PIASy (1 μg), as indicated. (C) PIASy represses TCRα enhancer activity. 293T cells were transfected with 0.25 μg of a TCRα-CAT reporter construct together with expression constructs encoding for β-galactosidase (50 ng; for normalization), LEF1 (100 ng), Ets1, and AML1 (250 ng each) or T7-PIASy (0.3 or 1 μg), as indicated. (D) Wild-type PIASy represses endogenous LEF1 activity. Jurkat cells were transfected with 1 μg of a LEF1 luciferase reporter construct containing multimerized LEF1-binding sites together with expression constructs encoding for β-galactosidase (0.5 μg; for normalization), β-catenin (5 μg), or increasing amounts of wild-type T7-PIASy (1 or 3 μg), RING-mutated T7-PIASy (1 or 3 μg), or Ser-mutated T7-PIASy (1 or 3 μg), as indicated. (E) Mutation of the SUMO consensus sites in LEF1 does not abrogate PIASy-mediated repression of LEF1 activity. 293T cells were transfected with 1 μg of a LEF1 luciferase reporter construct containing multimerized LEF1-binding sites together with expression constructs encoding for β-galactosidase (25 ng; for normalization), wild-type or mutant M2/K267R LEF1 (30 ng), β-catenin (1 μg), or increasing amounts of T7-PIASy (0.1, 0.3, or 1 μg), as indicated.