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. 2005 Feb;25(4):1475–1488. doi: 10.1128/MCB.25.4.1475-1488.2005

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Copyright © 2005, American Society for Microbiology

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FIG. 3. — Smad7 is required for the TGF-β-induced redistribution of β-catenin to the perinuclear-nuclear compartment. (A) Subcellular localization of endogenous Smad7 and β-catenin in PC-3U or PC-3U/AS-S7 cells after treatment with TGF-β for 2 h, 6 h, or 12 h as investigated by immunofluorescence with antibodies against Smad7 and β-catenin, together with additional staining of the nuclei by DAPI. Note that β-catenin is localized in the cytoplasm and in cell-cell contacts in untreated cells. After 2 h of TGF-β treatment, a portion of Smad7 is perinuclear or nuclear as β-catenin. At longer time points (12 h) after TGF-β treatment, both proteins predominantly localize in the perinuclear and nuclear compartment (left panel). In contrast, in PC-3U/AS-S7 cells, where the levels of endogenous Smad7 is reduced, no significant change of the subcellular localization of β-catenin was observed (right panel). (B) Quantification of the effect of TGF-β treatment on the subcellular localization of endogenous β-catenin in PC-3U and PC-3U/AS-S7 cells is shown in Fig. 2A. (C) PC-3U cells were transiently transfected with control (C) or specific Smad7 (S7) siRNAs and treated or not with TGF-β for the indicated times. The subcellular localizations of β-catenin and Smad7 were analyzed as described above. The values for the quantification of the subcellular localization of β-catenin in the transfected cells are shown in panel D. (F) HaCaT cells were transiently transfected with control (C) or specific Smad7 (S7) siRNAs and treated or not for the indicated time periods. The distribution of endogenous β-catenin and Smad7 was investigated by immunofluorescence and shown in panel E. The quantification of the differences in distribution of β-catenin is shown in panel F. Approximately 300 cells from each condition were counted under the microscope at 40× magnification. (G) Immunoblots from subcellular fractions of PC-3U and HaCat; nucleus (N) and cytoplasm (CP), derived from cells treated or not with TGF-β for the indicated time periods. Lamin A and β-tubulin were used as markers for the nucleus and cytoplasmic fractions, respectively. Total cell lysate from cells ectopically expressing Flag-Smad7 (PC-3U/pMEP4-Flag-S7) were used as the positive control for Smad7.

FIG. 3. — Smad7 is required for the TGF-β-induced redistribution of β-catenin to the perinuclear-nuclear compartment. (A) Subcellular localization of endogenous Smad7 and β-catenin in PC-3U or PC-3U/AS-S7 cells after treatment with TGF-β for 2 h, 6 h, or 12 h as investigated by immunofluorescence with antibodies against Smad7 and β-catenin, together with additional staining of the nuclei by DAPI. Note that β-catenin is localized in the cytoplasm and in cell-cell contacts in untreated cells. After 2 h of TGF-β treatment, a portion of Smad7 is perinuclear or nuclear as β-catenin. At longer time points (12 h) after TGF-β treatment, both proteins predominantly localize in the perinuclear and nuclear compartment (left panel). In contrast, in PC-3U/AS-S7 cells, where the levels of endogenous Smad7 is reduced, no significant change of the subcellular localization of β-catenin was observed (right panel). (B) Quantification of the effect of TGF-β treatment on the subcellular localization of endogenous β-catenin in PC-3U and PC-3U/AS-S7 cells is shown in Fig. 2A. (C) PC-3U cells were transiently transfected with control (C) or specific Smad7 (S7) siRNAs and treated or not with TGF-β for the indicated times. The subcellular localizations of β-catenin and Smad7 were analyzed as described above. The values for the quantification of the subcellular localization of β-catenin in the transfected cells are shown in panel D. (F) HaCaT cells were transiently transfected with control (C) or specific Smad7 (S7) siRNAs and treated or not for the indicated time periods. The distribution of endogenous β-catenin and Smad7 was investigated by immunofluorescence and shown in panel E. The quantification of the differences in distribution of β-catenin is shown in panel F. Approximately 300 cells from each condition were counted under the microscope at 40× magnification. (G) Immunoblots from subcellular fractions of PC-3U and HaCat; nucleus (N) and cytoplasm (CP), derived from cells treated or not with TGF-β for the indicated time periods. Lamin A and β-tubulin were used as markers for the nucleus and cytoplasmic fractions, respectively. Total cell lysate from cells ectopically expressing Flag-Smad7 (PC-3U/pMEP4-Flag-S7) were used as the positive control for Smad7.