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Fig. 4. Alterations in the localization, expression levels and charge of E-cadherin in SW480APC cells. (A) Redistrubution of E-cadherin in SW480APC cells. Cells were immunostained using an antibody against E-cadherin and Alexa488-conjugated anti-mouse IgG. Confocal microscopy reveals a punctate distribution for E-cadherin in SW480 and SW480 control cells (indicated by arrowheads) that is redistributed to sites of cell-cell contact in SW480APC cells (arrows). Bars, 10 µm. (B) Increased expression of E-cadherin in SW480APC cells by western blot analysis. Cells were lysed, 20 µg protein separated by 4-20% SDS-PAGE and immunoblotted using anti-E-cadherin and anti-actin (lower panel) mAbs. The level of E-cadherin in each clone was quantitated by densitometry, normalized to actin, and is represented as a ratio to E-cadherin in SW480 cells. Shown is a representative of at least three independent experiments. (C) 2DE gel analysis. SW480 or SW480APC.15 cells were lysed in 2DE gel extraction buffer containing 9 M urea, 4% (v/v) Chaps, 50 mM DTT, separated in two dimensions (IEF and SDS-PAGE), and analysed by immunoblotting with an anti-E-cadherin antibody. A paired sample of a representative of at least three independent experiments is shown.
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