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Fig. 5. E-cadherin prevents β-catenin and NF-
B nuclear localisation. (A) Cell fractionation of p65 and β-catenin. Subcellular fractions were prepared from SW-480 cells and analysed by western blot. Lamin B1 and TBP expression were used as nuclear markers; pyruvate kinase and E-cadherin were used as markers for the cytosolic-plus-membrane fraction. (B) Immunolocalisation of p65 and β-catenin is affected by E-cadherin expression. Analysis of β-catenin (green) and NF-B (red) subcellular localisation was carried out in SW-480 Snail1-transfected and SW-480 Snail1- and E-cadherin-transfected cells. E-cadherin-positive cells (right) were grown at equal cell density to E-cadherin-negative cells (left), or to a lower density (middle) in order to better visualize cell colonies. The analysis was performed by immunofluorescence using mAbs against β-catenin and NF-B. No signal was obtained when the same analysis was performed in the absence of primary antibody. (C) Subcellular co-distribution of p65 and E-cadherin. The subcellular distribution of NF-B p65 subunit (green) and E-cadherin (red) was determined by immunofluorescence in SW-480 E-cadherin-transfected cells as mentioned above, using specific mAbs against these two proteins. The upper row shows an amplified area selected from the panels shown below (boxed). An xz section is shown in the bottom row.
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