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Fig. 3. The effects of cytoskeleton-disruptive agents on the conversion of p115HEF1 to p105HEF1. A1F cells were serum-starved overnight, detached and resuspended in DMEM containing 0.1% heat-inactivated BSA and 2.5 ng/ml TGF-ß1. (A) Suspended cells were then plated onto FN-coated dishes and incubated for 4 hours. They were then treated for 2 hours with the indicated concentrations of nocodazole, colchicine, cytochalasin D (Cyto D) or latrunculin A. Cells treated with vehicle only were set as control (0). Cell lysates were generated and processed for immunoblotting with anti-HEF1 pAb. Nitrocellulose membranes were stripped and reprobed with anti-p130Cas mAb as loading control. (B) Suspended cells were seeded onto FN-coated coverslips for 4 hours and treated for 2 hours with the indicated concentration of nocodazole, cytochalasin D or the same volume of vehicle as control. Cells were then triple-stained for F-actin, microtubules and vimentin. Actin was visualized using Texas-Red-conjugated phalloidin (red); microtubules were visualized by staining the cell layers with anti-ß-tubulin mAb followed by the Alexa Fluor-350-conjugated secondary antibody (blue); and intermediate filaments were stained with anti-vimentin pAb followed by Alexa Fluor-488-conjugated secondary antibody (green).
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