The combination of perifosine with bortezomib-induced cytotoxicity is enhanced in combination with the anti-CD20 monoclonal antibody, rituximab. (A,B) BCWM.1 cells were cultured with the combination of perifosine (10 μM) and bortezomib (10 nM) for 4 to 6 hours and with rituximab (10 μg/mL) during the last hour. (A) NF-κB activity assay using Active Motif. NF-κBp65 transcription factor-binding to its consensus sequence on the plate-bound oligonucleotide was studied from nuclear extracts. WT and Mut are wild-type and mutated consensus competitor oligonucleotides, respectively. (B) Relative quantitative PCR of IκB gene. (C) Whole cell lysates were subjected to Western blotting using anti–p-Akt, -Akt, –p-S6R, and –α-tubulin antibodies. (D) BCWM.1 cells were treated similarly to that in panel A, and then whole cell lysates were immunoprecipitated overnight with anti-Akt antibody. Then the immunoprecipitates were subjected to in vitro kinase assay according to the manufacturer's protocol. Western blotting used -Akt and fusion protein -p-GSK3α/β antibodies. (E) Antibody-dependent cell-mediated cytotoxicity assay (ADCC). BCWM.1 cells were pretreated with either perifosine (10 μM), bortezomib (10 nM), and the combination overnight then washed then treated with rituximab (10 μg/mL) for 4 hours in the presence of the effector cells. Results are reported in terms of mean percentage of specific lysis characterized by measurement of release of calcein-AM with different effector:target ratios (E/T ratio). Data represent mean plus or minus SD of triplicate experiments (A,B,E).