As controls, aliquots of whole CHO lysates (inputs) and immunoprecipitations performed on CHO lysates using mouse non-immune IgG (IP Ctrl) were analyzed in parallel. A 110 kDa band corresponding to GST-FERM-DSR was detected in the anti-VE-Cad immunoprecipitate, indicating that GST- FERM-DSR is able to interact with VE-Cad. The anti-GST antibody recognized as in Fig 13 B GST-DSR and GST-FERM-DSR as well as an unspecific protein present in CHO cell lysates (star) Molecular weight markers are given at the left margin. (D) Cellular VE-Cad sequestration by MyoX FERM domain. VE-Cad-expressing CHO cells (40) were transiently transfected with plasmids coding for GFP-MyoX and either GST-DSR or GST- FERM-DSR. After cell lysis, Anti-MyoX immunoprecipitations were resolved on 4-12% gradient gels, electrotransferred and probed successively for VE-Cad, MyoX and GST. As controls, aliquots of the various CHO lysates and immunoprecipitations performed using rabbit non-immune IgG (IP Ctr) were analyzed in parallel. Molecular markers are given at the margin of each panel ,
Subconfluent HUVECs transiently co-transfected with plasmids expressing VE-CFP and GST-FERM-DSR were observed 20 h post transfection in phase contrast (I) and in the cyan and red fluorescence channelsIV) and VE-CFP and phase contrast merging (V, VI and VII) allowed the visualisation of two adjacent differentially-transfected cells: the cell at the right (delimited by yellow dotted line) co-expressed GST-FERM- DSR and VE-CFP while the cell at the left (delimited by pink dotted line) only expressed VE-CFP. Other cells of the fields are untransfected. The selected enlargements show that VE-CFP patches are at the cell-celljunctions (white arrows) and dispersed at the cell surface (white arrow-heads) in monotransfected cell (VIdotted rectangle in V) whereas in the double transfected cell, VE-CFP and GST-FERM-DSR co-localized and gathered around the cell nucleus (black arrow-heads, VII-rectangle in V) indicating that GST-FERM-DSR blocked the VE-Cad transport to the cell edge. In control experiments, in cells co-expressing GST-DSR and VE- CFP, VE-CFP patches are located at cell-cell-junctions (data not shown) Bars = 40 µm (I to V); 10 µm (VI and VII). (B) Blockage of intercellular contact formation by GST-FERM-DSR expression. The sequences are selected from Movie 9 and focus on the edges of two adjacent cells, the right column, the cell below expressed GST-FERM-DSR while the cell at the top is untransfected. In the left column, both cells expressed GST-DSR. Arrow-heads show unstable junctions whereas arrows point out stable junctions. Bars = 10 µm ,
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