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Figure 2


Fig. 2. BSG expression in cultured tumor cell lines MiaPaCa2, SW850 and Panc1. (A) Immunofluorescence staining of BSG: the human pancreatic adenocarcinoma cell lines were immunostained for BSG. Immunoreactivity was detected by fluorescein isothiocyanate (FITC) (green). Control was performed by omission of the primary antibody. Initial magnification was 200x. (B) Western-blot analysis of RIPA lysates and supernatant of MiaPaCa2, SW850, Panc1 cells as well as PSC. A band of 40-50 kDa according to the molecular mass of HG BSG was detected in supernatants as well as lysates. The LG form of BSG appeared as a band of 30-35 kDa in tumor cell lysates. Highly glycosylated proteins do not focus if separated by SDS-PAGE and glycosylation differs among the different cell types. Supernatants were concentrated 10-fold before western blot analysis. Lysates and supernatants were loaded adjusted to their protein concentration or adjusted to the surface area of the confluent well. Control was performed by omission of the primary antibody. (C) BSG immunofluorescence staining (FITC, green) of cancer cells co-cultured with PSC. Compared with cancer cells, the intensity of the BSG fluorescence was much lower on PSC as the result of a lower density of BSG in the cell membrane of PSC. The large PSC with their irregular polygonal fibroblast-like shape can be distinguished from the small round cancer cells in the phase-contrast image. Nuclei were counterstained with propidium iodide (red). (D) Agarose gel electrophoresis demonstrating the qRT-PCR products of BSG with a predicted length of 120 bp that were reverse transcribed and amplified from RNA of cultured MiaPaCa2, SW850 and Panc1 carcinoma cell lines as well as PSC (left panel). Controls comprised substitution of water for either cDNA (no template) or reverse transcriptase (no RT). In addition, the results of qRT-PCR were expressed normalized using XS13 as a housekeeping gene (right panel). The diagram is representative for three independent experiments.





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