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. 2007 Nov 5;28(2):825–835. doi: 10.1128/MCB.02375-06

FIG. 6.

FIG. 6.

Effect of C-terminally deleted mutant β-catenin and C-terminally deleted mutant plakoglobin and full-length plakoglobin on LEF/TCF-dependent transcription activated with stabilized mutant β-catenin. BPD cells were transiently cotransfected with pTOPflash and pCMV-renilla luciferase reporter plasmids and, where indicated, with expression vectors for stabilized mutant β-catenin (mβ), plakoglobin (1234), C-terminally deleted mutant β-catenin (ABC), or plakoglobin (123). A 0.1-μg amount of plasmid for mβ (0.1mβ) and 0.5 μg of plasmid for 1234, ABC, and 123 were transfected as indicated. Total plasmid DNA was adjusted to 1.1 μg by the addition of GFP expression vector, if necessary. (A) Expression of stabilized mutant β-catenin in BPD cells. Cells were transfected as described above. Western blots of total cell lysate were probed with anti-β-catenin antibodies. Since the epitope recognized by anti-β-catenin is in the C-terminal domain of β-catenin, anti-β-catenin antibodies do not recognize ABC. Note that the expression level of stabilized mutant β-catenin is not significantly affected by the expression of ABC. (B) Effect of C-terminally deleted mutants on TOPflash luciferase reporter activity. Cells were transiently transfected as described above. The value for BPD cells transfected with stabilized mutant β-catenin and GFP (0.1mβ+GFP) was set at 100. Note that transcriptional activity of stabilized mutant β-catenin is suppressed by cotransfection of C-terminally deleted mutant β-catenin (0.1mβ+ABC) but not plakoglobin and its C-terminal deletion (0.1mβ+1234 and 0.1mβ+123). The expression of C-terminally deleted mutants by themselves showed no transcriptional activity (ABC and 123). Error bars indicate standard deviations. (C) Subcellular localization of stabilized mutant β-catenin in BPD cells transfected as described above. At 20 h after transfection, cells were stained with anti-β-catenin MAb. Nuclei were visualized with DAPI. Note that cotransfection of C-terminally deleted mutant β-catenin did not affect nuclear localization of stabilized mutant β-catenin (0.1mβ+ABC). Bar, 20 μm.