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Figure 5


Fig. 5. Loss of E-cadherin in II-4 cells is linked to increased expression of {alpha}2, {alpha}3 and ß1 intregrin subunits. (A) Expression levels of integrin subunits on the cell surface of pBabe-, H-2kd-EcadC25- or H-2kd-Ecad-II-4 in 2D cultures. The three cell types were extracted and 5 µg samples of membrane proteins were immunoblotted in non-reducing conditions with anti-{alpha}2, anti-{alpha}3 or anti-ß1 integrin antibodies and analyzed by ECL. Expression levels of these specific integrin subunits were markedly increased in E-cadherin deficient H-2kd-Ecad-II-4 cells, when compared with their levels in the control pBabe- and H-2kd-EcadC25-II-4 cells (left panel). Scanning densitometry of the relative intensity of the presented immunoblots is shown on the right. (B) FACS analysis of pBabe-, H-2kd-EcadC25- or H-2kd-Ecad-II-4 cells immunoreacted with antibodies against specific integrin subunits. Elevated levels of cell surface {alpha}2, {alpha}3 and ß1 subunits in H-2kd-Ecad-II-4 cells (black line in right-hand panels) in comparison to control pBabe-II-4 cells (gray line) were identified by increased fluorescence intensity seen in E-cadherin deficient cells. The expression levels of {alpha}2, {alpha}3 and ß1 subunits were similar in H-2kd-EcadC25-II-4 (black line in left-hand panels) and the control pBabe-II-4 cells (gray line) as seen by the superimposition of these lines. (C) Functional blocking of ß1 integrin in H-2kd-Ecad-II-4 cells. Two-dimensional cultures of Ecad-deficient H-2kd-Ecad-II-4 cells were trypsinized briefly and replated for 15 minutes onto Type I and IV collagens or laminin-1 substrates, in the presence or absence of ß1-integrin-blocking antibodies. Attached cells were quantified by optical density determined at 590 nm. Results are calculated as the mean ± s.d. of four replicates and experiments were repeated three times. P<0.001 for cell adherence to Type I collagen, Type IV collagen and laminin-1 substrates compared with levels in blocked controls.





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