Synergistic apoptosis is associated with down-regulation of p-AKT in cell lines and primary human myeloma cells dependent upon order of addition and can be mimicked with the use of other AKT-specific inhibitors. (A) MM.1R cells were concurrently treated with bortezomib (8 nM or 20 nM) and lonafarnib (5 μM). After 24 hours, the cells were collected, and Western blots were performed with phos-AKT⁴⁷³ antibody. (B) RPMI8226 cells were concurrently treated with bortezomib (8 nM or 20 nM) and lonafarnib (5 μM). After 15 and 30 hours, the cells were collected, and Western blots were performed with Phos-AKT⁴⁷³ antibody in order to determine the time course for p-AKT reduction. (C) Kinase assay for AKT was performed using GSK α and β as a substrate. (D) Primary human MM cells were treated for 18 hours with either single agent or the combination of bortezomib and lonafarnib, and p-AKT or total AKT expression was analyzed using Western blot analysis. (E) MM.1R cells were concurrently treated with bortezomib (8 nM or 20 nM) and lonafarnib (5 μM) or LY294002 (20 μM) for 24 hours. Cells were harvested and stained with annexin V and PI as described in “Materials and methods.” Similarly, MM.1R cells were treated with AKT (1 and 2) siRNA, and after 24 hours, cells were collected, washed, and treated with bortezomib and lonafarnib for another 24 hours.