Figure 3. Vinculin closely interacts with the E-cadherin complex. (A) Recruitment of vinculin but not pY118-paxillin to cell–cell junctions in HGF-stimulated cells shown by IF. (B) Colocalization of vinculin but not paxillin with GFP–E-cadherin in E-cadherin–COMP adhesions revealed by IF. (C) Western blot (WB) analyses of total lysates (TL) and IP of endogenous E-cadherin, vinculin, paxillin, and β-catenin co-IP with GFP-tagged vinculin or E-cadherin from cell lysates after cross-linking. Black lines indicate that intervening lanes have been spliced out. (D, left) FRET from immunolabeled GFP-vinculin (Alexa Fluor 488) to immunolabeled β-catenin (β-ctn) or occludin (rhodamine). Error bars represent SEM (n = 18). (right) Mean acceptor fluorescence intensity in the ROIs used for calculating FRET. (E) MDCK cells transfected with GFP-vinculin or GFP–vinculin A50I were washed with CSK buffer, fixed, and immunostained for vinculin and GFP simultaneously.
Figure 3.

Vinculin closely interacts with the E-cadherin complex. (A) Recruitment of vinculin but not pY118-paxillin to cell–cell junctions in HGF-stimulated cells shown by IF. (B) Colocalization of vinculin but not paxillin with GFP–E-cadherin in E-cadherin–COMP adhesions revealed by IF. (C) Western blot (WB) analyses of total lysates (TL) and IP of endogenous E-cadherin, vinculin, paxillin, and β-catenin co-IP with GFP-tagged vinculin or E-cadherin from cell lysates after cross-linking. Black lines indicate that intervening lanes have been spliced out. (D, left) FRET from immunolabeled GFP-vinculin (Alexa Fluor 488) to immunolabeled β-catenin (β-ctn) or occludin (rhodamine). Error bars represent SEM (n = 18). (right) Mean acceptor fluorescence intensity in the ROIs used for calculating FRET. (E) MDCK cells transfected with GFP-vinculin or GFP–vinculin A50I were washed with CSK buffer, fixed, and immunostained for vinculin and GFP simultaneously.

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