Effect of LBH on upstream proteins involved in Akt activation. (A) After DHL-6 cells were treated with the indicated doses of LBH for 24 hours, cell lysates were immunoblotted for p-p85 and p-PDK1. (B) Immunoprecipitation of PP1 in lysates of LBH-treated cells, followed by immunoblotting with Akt. Simultaneously LBH-treated whole-cell lysates (WCLs) were immunoblotted with PP1 and PP1A. (C) Effect of protein phosphatase inhibitor calyculin A on LBH-induced Akt dephosphorylation. DHL-6 cells were exposed to LBH and calyculin A (50 nM) for 24 hours and cell lysates were immunoblotted. (D) Repressed expression of HDAC3, but not HDAC4, led to Akt dephosphorylation in a manner similar to that of LBH treatment. The cell lysates were immunoblotted with antibodies against phospho-Akt. (E) DHL-6 cells were transiently transfected with CA-Akt and empty vector, and subsequently treated with the combination for 48 hours, and survival was assessed. Bar graph shows mean ± SD. (F) CA-Akt–transfected cells were analyzed for Akt activation using p-Akt antibody. (G-H) Effect of LBH on rictor/raptor complex. Ly7 cells were treated with rapamycin (10 nM) or LBH (50 nM) or the combination for 24 hours. WCLs and mTOR immunoprecipitates prepared from the lysates were analyzed by Western blot for the levels of mTOR, rictor, and raptor.