Figure 4.

Plakoglobin (PG) expression reduces β‐catenin interaction with T‐cell factor 4 (TCF‐4), decreases β‐catenin/TCF‐4 reporter activity and target gene expression. A, Equal amounts of total cellular extracts (TCE) from H1299 cells and H1299 transfectants were processed for sequential immunoprecipitation (IP) and immunoblotting (IB) using plakoglobin, nuclear β‐catenin (β‐cat) and TCF‐4 (TCF) antibodies. Equal loadings were confirmed by processing total cell extracts from all cell lines for immunoblotting using β‐actin antibodies. B, Untransfected H1299 cells and H1299 transfectants were cotransfected with pTOPFLASH β‐catenin/TCF reporter construct and p‐RL‐TK Renilla reporter plasmid and luciferase activities were measured 48 h post‐transfection. Levels of luciferase activities from pTOPFLASH were normalized to those of Renilla plasmid. Four independent experiments were carried out and the results were normalized to H1299 parental cells. SW620 cells were included as a positive control. UT, untransfected. *P < .05, **P < .001. C, Total cellular RNA was extracted from untransfected H1299 cells and H1299 transfectants, reverse transcribed and processed for real‐time PCR for C‐MYC (top) and S100A4 (bottom) using specific primers (Table 2). Expression levels were first normalized to the amount of ACTB in the same cell line and then to H1299 untransfected cells. Histograms were constructed based on the average ± SD. UT, untransfected. *P < .05, **P < .001