Differential targeting of perifosine and bortezomib on Akt, Erk, and proteasome pathways. (A) BCWM.1 cells were cultured with the combination perifosine (10 μM) and bortezomib (10 nM) for 6 hours and rituximab (10 ng/mL) in the last hour, and then the effect on Akt and ERK MAPK pathways was assessed by immunobloting. Whole cell lysates were subjected to Western blotting using anti–p-Akt, -Akt, –p-GSK3α/β-p-S6R, -ERK1/2, –p-ERK, and –α-tubulin antibodies. (B) BCWM.1 cells were cultured with the combination perifosine (10 μM) and bortezomib (10 nM) for 6 hours, and then whole cell lysates were immunoprecipitated overnight with anti-Akt antibody. Then the immunoprecipitates were subjected to in vitro kinase assay according to manufacturer's protocol. Western blotting used -Akt and fusion protein -p-GSK3α/β antibodies. (C) BCWM.1 cells were cultured with either triciribine (5 μM and 10 μM), U0126 (5 nM and 10 μM), or the combination. Cytotoxicity was assessed using MTT assay. (D) 20S proteasome activity assay. BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (10 nM), or the combination for 4 and 12 hours, and then whole cell lysates were subjected to proteasome activity measurement. Data represent mean plus or minus SD of triplicate experiments.