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Figure 4


Fig. 4. Alterations in focal adhesion contacts in Cx43{alpha}1 KO and CMV43 CNCs. (A-C) CNC cells from wild-type (A), Cx43{alpha}1KO (B) and CMV43 (C) embryos were cultured on 15 µg/ml fibronectin matrix. Cells were double immunostained with antibodies against ß1 integrin (red) or vinculin (green). Cx43{alpha}1KO cells show reduced ß1 integrin and vinculin immunostaining (B). In CMV43 CNCs, the pattern of ß1 integrin and vinculin immunofluorescence is qualitatively changed, with the intensity of vinculin immunostaining showing a noticeable increase (C). Magnification is same in all panels. Scale bar: 50 µm. (D) To examine if ß1 integrin is expressed in CNCs, wild-type and CMV43 CNC explants were surface biotinylated, then cell extracts were made, followed by immunoprecipitation using a ß1 integrin antibody and western immunoblotting using stretpavidin-peroxidase. This yielded a 120 kDa band, the size expected for ß1 integrin. No difference was detected in the surface ß1 integrin expression level in the CMV43 and wild-type CNCs. (E,F) The mean area and mean intensity of vinculin immunofluorescence in CNCs cultured on 1, 15 and 50 µg/ml fibronectin matrix were quantitatively assessed. Cx43{alpha}1KO cells showed decreases in vinculin area and intensity (E), while CMV43 cells showed an increase in vinculin intensity (F). *P<0.05, **P<0.01; #P<0.0005. n/s, no significant difference, when compared with Cx43{alpha}1+/+ or nontransgenic cells.





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