|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 5. Cells adhere to secreted epimorphin and activate focal adhesion kinase. (A) SCp2 cells adhere to intact secretory protein complex containing 30 kDa soluble epimorphin, 40 kDa synaptotagmin and annexin II, accompanied by FAK activation. This activity is selectively blocked by anti-epimorphin antibodies. The immobilization procedure was carried out under physiological conditions using the T7-tag as the target site for the immobilization. Western blotting of the immobilized materials confirmed successful immobilization of 30 kDa epimorphin, annexin II and 40 kDa synaptotagmin after incubation with supernatant from TE transfectants but not from the control cells (Vector). The amount of captured epimorphin on the substrate was about 100 ng/cm2, as determined by a luminescent image analyzer Las1000plus using purified recombinant epimorphin as reference. The number of cells bound to each well in 4 hours was counted and the relative number to collagen-coated wells was calculated. Data are the mean ± s.d., n=4, *P<0.05. P-FAK, FAK phosphorylated at Y397. Bar, 50 µm. (B) Recombinant forms of secreted epimorphin r-Epm30 (aa 1-245) and r-Epm (aa 1-188), but not recombinant epimorphin SNARE domain r-SNARE (aa 189-265), bind to SCp2 cells with FAK/ERK activation. Images show cells starved for 24 hours, incubated with the indicated recombinant protein for 30 minutes; bound protein (red) and E-cadherin (green). Bars, 20 µm. Blots on the right show phosphorylation of FAK in cells treated with a recombinant protein for 15 and 30 minutes (first column of blots). phosphorylation of ERK but not EGF receptor was detected in cells treated with r-Epm for 30 minutes (second column of blots).
|
