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Fig. 4. Cortactin physically associates with, and is recruited to, nascent N-cadherin adhesions. (Ai) N-cadherin was immunoprecipitated from lysates of DAM using N-cadherin antibody (A, 15, 60, 180, 360, 720) or with an irrelevant, control antibody (U). Acceptor monolayer (A) was used as a baseline sample and donor-acceptor culture samples at 15, 60, 180, 360 and 720 minutes represent de novo N-cadherin-mediated contacts. Densitometric analysis of N-cadherin:cortactin ratios from immunoprecipitation timeline reveals dramatic cortactin recruitment within 15 minutes to the adhesion complex with a peak at 60 minutes followed by a decrease as junctions mature. (ii) Immunoprecipitation conducted using cortactin antibody and immunoblotted for N-cadherin confirm rapid association of cortactin with denovo contacts. (iii) Extent of N-cad-Fc recombinant protein bound to the surface of magnetic beads analyzed by immunoblotting. CM, Sup, Elution indicate starting conditioned media (15 µg/ml), supernatant of bead pull down, and elution from bead surface, respectively. Right panel: magnetic Ncad-Fc-coated beads used in bead pull-off assay to show cortactin association and recruitment to adhesion complex from within 15 minutes. The 15B sample is indicative of nonspecific protein association with the surface of bare beads following 15 minutes incubation. Equivalent amounts of protein were loaded as determined by BioRad Assay. (B) GFP-cortactin (full length) transfected donor cells were allowed to attach to an incompletely confluent layer of acceptor cells and processed for live videomicroscopy from initial attachment to 30 minutes. Three frames are shown showing cortactin distribution and recruitment to sites of donor-acceptor adhesion.
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