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

FIG. 2.

FIG. 2.

Smad7 is required for TGF-β-induced association between β-catenin and LEF1. (A) Lysates from PC-3U (upper panel) and HaCaT cells (lower panel), transiently transfected with control (C-siRNA) or specific Smad7 siRNAs (S7-siRNA), were treated or not with TGF-β for the indicated time periods and the total cell lysates were subjected to coimmunoprecipitation with a polyclonal antibody for β-catenin or a nonimmune, control (C) antibody and then analyzed for coprecipitated LEF1. Cell lysates from 293T cells transiently transfected with HA-LEF1 were used as a positive control (Pos.Ctrl.) to verify the identity of endogenous LEF1. The cell lysates were investigated for the amount of endogenous LEF1, β-catenin, and Smad7. The filters were then stripped, blocked, and reprobed with actin antibodies to confirm equal loading of proteins. (B) The same cell lysates from PC-3U and HaCaT cells were also investigated for the amount of endogenous Smad7 and c-Myc by immunoblotting. Total cell lysates from cells ectopically expressing Flag-Smad7 (PC-3U/pMEP4-Flag-S7) were used as positive control for Smad7 (Pos. Ctrl). The filters then were stripped, blocked, and reprobed with actin antibodies to confirm equal loading of proteins.