Figure 5: Activation of AKT is associated with increased XIAP protein expression and Apo2L/TRAIL resistance. (a) Western blot assays of VH-64 cell lysates with antibodies to phosphorylated AKT and total AKT. Cells were treated for the indicated times with IGF1 (100 ng/mL). The antitotal AKT blot serves as a loading control. Representative nonstripped blots separately probed with the different antibodies are shown. (b) Ratio of phosphorylated AKT to total AKT determined by ELISA in cells treated for the indicated times with IGF1 (100 ng/mL). (c) Percentage-specific apoptosis in VH-64 cells treated without (white columns) and with 50 nM (grey columns), 200 nM (striped columns), and 500 nM (black columns) wortmannin in the absence and presence of IGF1 (50 ng/mL; 24 hours), followed by incubation without and with Apo2L/TRAIL (TRAIL; 50 ng/mL; 8 hours). (d) EIA analysis of XIAP in cells treated with wortmannin (WM; 500 nM), AKTi-1/2 (2 μM), and serum-free medium alone (Control), followed by incubation in the absence and presence of IGF1 (100 ng/mL; 24 hours). Asterisks: *, **; error bars: mean ± SD of triplicate determinations from 3 independent experiments.