FIG. 5.

Dominant-negative AKT blocks the antianoikis effect of MSP. (A) RE7 cells were transiently transfected with kinase-dead AKT (d/n AKT) or with the pCMV vector as a control. Anoikis was induced as described in the legend to Fig. 1, and DNA fragmentation in apoptotic cells was quantified by cell death ELISA. Three independent experiments were performed. The results of a representative experiment are shown. The error bars indicate the standard deviations for triplicate ELISA wells. (B) Expression of dominant-negative AKT blocks MSP-induced endogenous AKT enzymatic activity. RE7 cells, transiently transfected with the empty vector (pCMV) or dominant-negative AKT cDNA, were stimulated with 5 nM MSP for 15 min. The cells were then lysed, and AKT was immunoprecipitated and tested for kinase activity in vitro. The kinase assay was performed as described in the legend to Fig. 4A. Incorporation of ³²P into AKT was detected by SDS-PAGE and autoradiography.