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Figure 5


Fig. 5. Nectin-3- and afadin-mediated regulation of PI3K activity in NIH3T3 cells. (A) Inhibition of the PDGF-induced increase in PI3K activity by knockdown of nectin-3 and afadin in NIH3T3 cells. After 16 hours of serum starvation, control, nectin-3-knockdown and afadin-knockdown NIH3T3 cells were treated with 3 ng/ml PDGF for 2 minutes. Lysates from cells harvested before and after PDGF treatment were subjected to an assay for PI3K activity. Bars in the graph represent the relative spot intensity compared with the values of control NIH3T3 cells treated with PDGF, which are expressed as 1. KD, knockdown; PI(3)P, phosphatidylinositol 3-phosphate. (B) Association of FLAG-afadin with PI3K. Untransfected HEK293 cells and HEK293 cells transiently transfected with FLAG-afadin were lysed and the cell lysates were immunoprecipitated with the anti-FLAG mAb or the anti-p85 pAb. The immunoprecipitates were subjected to western blotting with the anti-p85 pAb and the anti-FLAG mAb. (C) Association of endogenous afadin and PI3K in NIH3T3 cells. Lysates of NIH3T3 cells were immunoprecipitated with the anti-p85 pAb or non-immunized rabbit IgG as a control, and the immunoprecipitates were subjected to western blotting with the anti-afadin mAb and the anti-p85 pAb. The results shown in this figure are representative of three independent experiments.





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