(A) Western blotting for basal protein abundance of REST, p-AKTSer473, total AKT, and histone H3 (control) in DAOY, UW426 and UW228 cells. Representative blots are shown; long/short indicate exposure times. (B and C) Western blotting for total and phosphorylated (p-AKTSer473) protein abundance after either (B) shRNA-mediated REST knockdown in UW228 and DAOY cells or (C) REST overexpression in DAOY cells. Blots are representative images of 3 experiments. (D) MTT assay-derived proliferation of UW228 and DAOY cells treated with various doses of MK2206 for 24, 48 or 72 hours. Data are means ± S.D. of 3 independent assays. (E) Western blotting for abundance of p-AKTSer473, total AKT, cleaved Caspase-3, cleaved PARP and histone H3 (control) to assess induction of apoptosis following treatment of UW228 cells with MK2206 (5 μM) for 12 or 24 hours. Representative blots are shown (n=3).