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Fig. 5. p120 is necessary for both cell spreading on Ncad-Fc substrate and cadherin adhesion formation. (A) C2 cells were transfected either with siRNA p120-pSUPER or the empty pSUPER vectors, 16 hours prior to protein extraction. Twenty µg of protein extracts were separated on 7% SDS-PAGE and analysed by immunoblotting with anti-p120 and anti-ß-catenin antibodies. Tubulin was used as a loading control. (B) p120-pSUPER-transfected cells and mock transfectants were fixed and immunostained for p120 and ß-catenin. Note the very low level of p120 immunostaining in p120-pSUPER-transfected cells and the remaining accumulation of ß-catenin at cell-cell contacts (arrows). (C) C2 cells double transfected with pEGFP and either p120-pSUPER or empty-pSUPER were plated on Ncad-Fc substrate for 2 hours and labelled with anti-ß-catenin antibodies or Alexa-conjugated phalloidin. Note the absence of spreading of p120-pSUPER-transfected cells, contrasting with the normal spreading and cadherin adhesion formation of mock transfected cells. (D) C2 cells double transfected with the p120-pSUPER and DA Rac1-GFP were plated on Ncad-Fc and stained for ß-catenin or actin. Notice the restored spreading of the cells. Bars, 10 µm.
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