Electromobility shift assays www.selleckchem.com/products/arq-197.html (EMSA) Nuclear extracts, HTF/AP-2 cis sequence and EMSA were described elsewhere (Schreiber et al, 1989; Vernimmen et al, 2003). Briefly, 2�C4��g of crude nuclear proteins were incubated with 300000c.p.m. of [��-32P]dCTP end-labelled oligonucleotide. The retarded complexes were analysed on a nondenaturing 5% polyacrylamide gel and analysed using a PhosphorImager (Molecular Dynamics Amersham Biosciences, Roosendal, The Netherlands). Western blotting For p185c-erbB-2 detection, cells were scraped off the culture dishes, harvested in PBS, pelleted by centrifugation, resuspended in a 1% SDS solution and boiled for 10min. Whole cell extracts (20��g) were loaded per well, separated on a 12% SDS�Cpolyacrylamide gel and transferred to a PVDF membrane (Millipore, Brussels, Belgium).
A c-erbB-2 antibody (06-562 Euromedex, Mundolsheim, France) was used at a 1:2000 dilution. For AP-2 detection, 10�C25��g of nuclear extracts were loaded per well. An AP-2�� antibody (sc-184 Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a 1:700 dilution. Secondary antibodies (Dako Diagnostics, Glostrup, Denmark) were detected with the ECL system (Amersham BioSciences). The ��-actin monoclonal antibody was from Sigma (monoclonal (amoeba) mouse ascites fluid clone KJ43A Sigma-Aldrich Bernem, Belgium). Plasmids and transient transfection assays The transfection efficiencies of all the cell lines were tested by transfection of the pEGFP-IRESpuro control vector (Clontech, Palo Alto, CA, USA). Cells were transfected using the FuGENE 6 reagent (Roche).
Cells (4 �� 105) were plated on 35mm tissue culture dishes with a FuGENE/DNA ratio of 3:1. The cells were incubated for 48h in complete medium. Cells transfected with the green fluorescent protein (GFP) expression plasmid were visualised by fluorescent microscopy. The luciferase (LUC) reporter vectors containing different ERBB2 promoter fragments have been previously described (Grooteclaes et al, 1994). The LUC enzymatic activities were measured using the Luciferase Reporter Gene Assay kit (Roche). RESULTS ErbB-2 gene copy number, mRNA and protein levels in cancer cell lines We measured the p185c-erbB-2 protein levels by ICC and Western blotting. For ICC, we used the well characterised breast cancer cell lines BT-474 and ZR-75-1 as standards to determine the erbB2 expression in the non-breast cells (Figure 1A, C).
After ICC, p185c-erbB-2 appeared as a brown membrane staining in positive breast Dacomitinib cancer cells. Likewise, p185c-erbB-2 exhibited an intense membranous staining in SK-OV-3 ovary carcinoma cells (Figure 1B). P185c-erbB-2 was detected in one out of two ovary, four out of five colorectal, three out of three prostatic and only two out of seven pancreatic cancer cells, but the staining was heterogeneous and mainly cytoplasmic (Figure 1D and Table 1). P185c-erbB-2 was also detected in the cytoplasm of HepG2 hepatocarcinoma cells (Figure 1E).