Perifosine enhances cytotoxicity of novel (bortezomib) agents. (A) MM.1S cells were cultured for 24 hours with control media (□) and with 2.5 μM (▦) or 5 μM (▪) perifosine in the presence or absence of bortezomib (5 nM and 7.5 nM). Cytotoxicity was assessed by MTT assay; data represent means (± SD) of quadruplicate cultures. (B) MM.1S cells were cultured with bortezomib (10 nM) for 4 hours and 8 hours. Whole-cell lysates were subjected to Western blotting with p-Akt and Akt Abs. (C) MM.1S cells were cultured with bortezomib (10 nM) for 8 hours. Whole-cell lysates were immunoprecipitated with anti-Akt Ab. The immunoprecipitates were washed and subjected to in vitro kinase assay, according to manufacturer's protocol. The reaction mixtures were immunoblotted with anti–p-GSK3α/β. (D) MM.1S cells were cultured with bortezomib (5, 10, and 20 nM) in the presence (5 μM) or absence of perifosine. Whole-cell lysates were subjected to Western blotting with p-Akt, Akt, p-ERK, and ERK2 Abs. (E) MM.1S cells were cultured for 8 hours with control media, perifosine (5 μM), bortezomib (10 nM), or perifosine (5 μM) plus Bortezomib (10 nM). Cells were then lysed and subjected to Western blotting using anti–p-JNK1/2, caspase-8, and PARP Abs.