Enzastaurin targets the Akt signaling pathway. (A) BCWM.1 cells were cultured with control media or enzastaurin (2.5 to 10 μM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–pan–p-PKC (ser660), anti–p-PKCβ (thr500), anti-PKCβ, and α-tubulin antibodies. (B) BCWM.1 cells were cultured with control media, enzastaurin (2.5 to 10 μM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-MARCKs, anti–p-GSK3α/β, anti–p-S6 ribosomal, and anti–α-tubulin antibodies. (C) BCWM.1 cells were cultured with enzastaurin 7.5 μM for the time indicated. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, total Akt, anti–p-GSK3α/β, anti–p-S6 ribosomal, and anti–α-tubulin antibodies. (D) Akt kinase assay. BCWM.1 cells were cultured with control media or enzastaurin 7.5 μM for 30 minutes or 1 hour. Whole-cell lysates were immunoprecipitated with anti-Akt antibody, washed, and subjected to in vitro kinase assay according to the manufacturer's protocol. Western blotting used Akt and phospho-GSK3α/β antibodies. (E) BCWM.1 cells were cultured with enzastaurin (2.5 to 10 μM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-ERK, anti-ERK1/2, and α-tubulin antibodies. (F) BCWM.1 cells were cultured for 48 hours with media and with enzastaurin (5 to 7.5 μM) in the absence or presence of 5 and 10 μM MEK1/2 inhibitor U0126. Cytotoxicity was assessed by MTT assay. Data represent mean (± SD) of triplicate experiments.