Fig. 4. Regulation of PI 3-kinase and JNK activities by cell adhesion to fibronectin, and the effect of PI 3-kinase inhibitor on the adhesion induction of Rac1 activation. 0.5% serum-starved CHO, ß1, ß3 and ß1-3-1 cells were detached and kept in suspension in DMEM for 1 hour and then plated on fibronectin-coated dishes for 4 hours. In A, cell lysates from the various samples following adhesion to fibronectin were immunoprecipitated with anti-PI 3-kinase p85 antibody followed by the PI 3-kinase activity assay. Shown at the bottom is the amount of PI 3-kinase p85 immunoprecipitated from 200 µg cell lysates, indicating that comparable amounts of PI 3-kinase p85 were immunoprecipitated in these samples. (B) The effect of a PI 3-kinase inhibitor, LY294002, on adhesion induction of Rac1 activity is shown in the top panel. The bottom immunoblot shows Rac1 in whole cell lysates. The amount of activated Rac1 was normalized by the amount of Rac1 in whole cell lysates. (C) The top panel shows JNK kinase activity using GST-c-Jun as the substrate, and the bottom panel is the immunoblot using anti-JNK1, which indicates equal loading. The bar graphs in A-C are the densitometric analyses, representing the mean±s.d. of three separate experiments. The asterisks in A and C indicate significant differences (P<0.05) compared with CHO cells, and the asterisk in B indicates a significant difference (P<0.05) compared with cells without the PI 3-kinase inhibitor.

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