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Fig. 2. LPA stimulation of adhesion movement. Rat2 or NIH3T3 cells stably expressing GFP-FAK or paxillin-GFP were plated in the presence of serum-containing (10%) medium on dishes coated with 1 µg/ml fibronectin. The following day, cells were placed in serum-free medium for 12 hours and subsequently stimulated with 2 µM LPA. (A) Analysis of adhesion movement in response to LPA. Serum starved NIH3T3 cells stably expressing paxillin-GFP were stimulated with 2 µM LPA and the movement of adhesions was analyzed using TIRF microscopy as described in Materials and Methods. Cells were filmed for 7 minutes before addition of LPA and 7 minutes after addition of LPA (arrows indicate representative adhesions). Adhesions present at time 0 were pseudo-colored green (left panels) and adhesions present at 7 minutes were colored red (middle panels). Movement of the individual adhesions was revealed using the merged images (right panels) and analyzed as described in Materials and Methods. (B) Quantification of adhesion movement by LPA in NIH3T3 cells expressing paxillin-GFP was determined as described in Materials and Methods. The experiment was performed three times, and five to seven cells were analyzed per experimental condition. (C) Western blot analysis of FAK activation by LPA in Rat2 cells stably expressing GFP-FAK. Cells were stimulated with LPA for 5 minutes and GFP-FAK phosphorylation was assessed using antibody against phosphorylated tyrosine (pTyr) as described in Materials and Methods. The increase in activation of MAP kinase was determined by immunoblotting using antibodies against phosphorylated and total ERK (pERK and ERK, respectively). (D) LPA activation of endogenous FAK in Rat2 cells. Cells were stimulated with LPA for 5 minutes and western blots were probed with antibody against phosphorylated Tyr397 (pY397) or anti-FAK antibody as described in Materials and Methods.
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