Figure 6
From: Beta-catenin cleavage enhances transcriptional activation
Overexpressed double truncated β-catenin increases transcriptional activity. (A) NCM460 cells were infected with FS, ∆∆ and ∆N142 β-catenin constructs tagged with His (N terminus) and Flag (C terminus). WBs were probed sequentially with anti-Flag and anti-His antibodies. Fibrillarin served as loading and purity controls for chromatin bound fractions. (B) Total cell lysates of NCM460 cells overexpressing FS β-catenin were precipitated with anti-Flag antibody or (Co2+) sepharose (His tag specific). Proteins were resolved on SDS PAGE and WBs probed sequentially with N and C termini specific anti-β-catenin antibodies. (C) Schematic representation of β-catenin constructs used in (A and B). (D) Chromatin bound fractions from NCM460 cells are shown: vector control, FS, ∆∆ and ∆N89 β-catenin overexpressing cell lines immunoprecipitated with anti-TCF4 and probed with pβ-Cat552 and core region antibodies. Fibrillarin serves as a loading control for input. (E) FS, ∆∆ and ∆N89 β-catenin overexpressing NCM460 cell lines were co-transfected with TCF/LEF reporter plasmid and β-catenin-induced luciferase activities measured. *p = 0.002, **p = 0.001, ***p = 0.003. Flag WB of total cell lysates used in (D and E) (to evaluate levels of overexpressed β-catenin) can be seen on Suppl. Fig. S8C. Full size membrane scans for WBs can be seen in Suppl. Fig. SS6.
