Perifosine affects membrane distribution of HA-Akt1 and Akt1-PH domain constructs. Perifosine affects membrane distribution of HA-Akt1 and Akt1-PH domain constructs. A, at 24 h post-transfection with the indicated construct, cells were starved for another 24 h and then treated with perifosine (5 μm) for 30 min followed by insulin (50 nm) stimulation for another 5 min. The cells were blocked and probed with FITC-conjugated HA as described in “Materials and Methods.” The cells were observed under 63× magnification using Olympus microscope. Red arrows, nontransfected cells and nuclear staining with 4′,6-diamidino-2-phenylindole. White arrowheads, membrane localization of Akt. B, subconfluent PC-3 cells grown on coverslips were transfected with pCEFL-EGFP-AH-Akt1. At 24 h post-transfection, cells were starved for another 24 h and then treated with perifosine (5.0 μm) for 30 min followed by induction with insulin (50 nm) for another 5 min. The cells were fixed and slides were prepared as described in “Materials and Methods.” GFP immunoflourescence images were monitored on PC-3 cells transfected with pCEFL-EGFP-AH-Akt1. The green fluorescence was observed under 63× magnification. Sudhir B. Kondapaka et al. Mol Cancer Ther 2003;2:1093-1103 ©2003 by American Association for Cancer Research