NVP-BEZ235 targets Akt and mTOR, and inhibits Akt and mTOR activity in WM cells and other low-grade lymphoma IgM-secreting cell lines. (A-B) BCWM.1 cells were cultured with NVP-BEZ235 (5-100nM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-S6R, anti–p-GSK3α/β, anti–p-mTOR, anti-mTOR, p-p70S6, anti–p-4EBP1, and anti–α-tubulin antibodies. (C) BCWM.1 cells were cultured with NVP-BEZ235 (20nM) or control medium for 6 hours. Immunocytochemical analysis was assessed using anti–p-Akt and p-mTOR antibodies. The 4′,6-diamidino-2-phenylindole was used to stain nuclei. (D) In vitro Akt and in vitro mTOR kinase assays. BCWM.1 cells were cultured with control media or NVP-BEZ235 (20-50nM) for 6 hours. Akt kinase assay: whole-cell lysates were immunoprecipitated with anti-Akt antibody. Then the immunoprecipitate was washed and subjected to in vitro kinase assay, according to the manufacturer's protocol. Western blotting used anti–p-GSK3α/β and anti-Akt antibodies. mTOR kinase assay: whole-cell lysates were immunoprecipitated with anti-mTOR antibody. Then the immunoprecipitate was washed and subjected to in vitro kinase assay, according to the manufacturer's protocol. Enzyme-linked immunosorbent assay–based assay has been performed (anti-p70S6K was added to each well), and absorbance read at 450nM with a reference wavelength set at 540nM. (E) IgM-secreting cell lines (MEC.1; RL) were cultured with NVP-BEZ235 (10-200nM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-S6R, anti–p-GSK3α/β, anti–p-mTOR, anti–p-4EBP1, anti-mTOR, and anti–α-tubulin antibodies.