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Fig. 9. Dynamic MTs are necessary for myosin II activation at cell-cell contacts. (A) Nocodazole blocks activation of MLC by E-cadherin homophilic ligation. Control MCF-7 cells or cells pretreated with nocodazole (100 nM, 60 minutes; noc) were allowed to adhere to hE/Fc- or poly-L-lysine (PLL)-coated substrata for 90 minutes before lysis. Western blots of cell lysates were probed for activation-specific phosphorylated MLC (ppMLC) or ß-tubulin as a loading control. (B,C) Nocodazole blocks accumulation of phosphorylated MLC at cell-cell contacts. Confluent MCF-7 monolayers were incubated with nocodazole (100 nM) for 1-3 hours, then processed and stained for phosphorylated MLC or E-cadherin. (B) Phosphorylated MLC staining was consistently observed at cadherin-based cell-cell contacts in control cells (arrowheads). By contrast, many contacts in nocodazole-treated cells showed little or no co-accumulation of phosphorylated MLC (arrows). (C) Co-accumulation of phosphorylated MLC at cadherin cell-cell contacts was quantitated by counting the number of cadherin-positive contacts that also showed phosphorylated MLC staining.
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