Protein quantification was performed using the BCA protein assay. Western blotting analyses were performed, read FAQ as previously described (Song et al, 2000, 2003a), using antibodies against ��-catenin (Transduction Laboratories, Lexington, KY, USA) and cyclin D1 (Pharmingen, Chicago, IL, USA). Ponceau S (Sigma, St Louis, MO, USA) staining and immunoblots with either a monoclonal ��-actin antibody (Sigma) for whole-cell lysates or polyclonal Sp1 antibody (Santa Cruz, Santa Cruz, CA, USA) for nuclear extracts were used to confirm equal loading of Western blot membranes. Reporter assays MC-26 cells (1�C2 �� 105) were plated 1 day prior to transfection. For transient transfection experiments, subconfluent cells were incubated with DNA and FUGENE-6 liposome reagent (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instructions.

Both LEF-1 and cyclin D1 (minimal and full-length cyclin D1 promoter-luciferase constructs; Albanese et al, 2003) experiments utilised the FUGENE-6 transfection reagent to deliver the target plasmids. In addition, renilla was cotransfected with the firefly reporter plasmids to normalise for transfection efficiency. Total DNA was balanced with the addition of empty vector when multiple plasmids were used. At 24h after the transfection, the cells were incubated with 20 and 50nM G-17 for 4h, harvested, and assayed for firefly luciferase reporter and for renilla activity. Briefly, 10��l of protein extract was first assayed with 50��l of firefly luciferase substrate (Promega, Madison, WI, USA) in a luminometer for 10s.

Subsequently, in the same tube, 50��l renilla substrate (Promega) were added and measured. Samples were assayed in duplicate, and the luciferase counts were normalised to renilla measurements. In vitro kinase assay Equal amounts of protein extracted from MC-26 cells (10��g) were assayed for CK2 kinase activity, as previously described (Song et al, 2000, 2003a). Briefly, each sample was assayed in duplicate with and without CK2-specific synthetic peptide, RRREEETEEE (Promega), for 20min at 37��C with 5��Ci [��-32P]ATP. Radioactive counts were blotted onto p81 filter circles, washed 4 �� in 150mM H3PO4, and analysed on an automated liquid scintillation counter. Buffer controls with and without the substrate peptide (background control) were also measured and subtracted from the final radioactive counts.

Statistical analysis The two-way Student’s t-test was performed for paired comparisons. Statistical significance was assigned if P<0.05. RESULTS Gastrin-17 enhances ��-catenin protein levels in MC-26 cells To determine the potential role of gastrin in modulating ��-catenin, mRNA and protein levels were measured by Northern and Western blot hybridisations, respectively, Brefeldin_A in MC-26 cells that were transiently treated with G-17.