7. Western blot analysis

Proteins were extracted, and equal amounts of protein were separated on 5%-15% sodium dodecyl sulfate–polyacrylamide gels as previously described [20]. Separated proteins were transferred onto nitrocellulose membranes, and the blots were probed with primary antibodies (1:1,000 dilution) overnight at 4°C. Antibodies specific for the following factors were used: Pim-1 (sc-13513, Santa Cruz Biology Technology, Santa Cruz, CA), Pim-2 (CST 4730, Cell Signaling Technology, Beverly, MA), Pim-3 (CST 4165), phosphorylated (p)Bad Ser112 (CST 9296), Bad (CST 9292), p4EBP1 Ser65 (CST 9451), 4EBP1 (CST 9452), pS6K (CST 2211), S6K (CST 2217), Bcl-xL (sc-1690), Mcl-1 (sc-819), PARP (BD 556-494), caspase-3 (CST 9662), light chain 3B (LC3B; CST 3868), Beclin-1 (ab51031), pATM Ser1981 (CST 4526), ATM (sc-239-21), pChk2 Thr68 (CST 2661), Chk2 (sc-5278), pAkt Ser473 (CST 9271), Akt (CST 9272), pPRAS40 Thr246 (CST 2997), PRAS40 (CST 2691), and α-tubulin (Sigma-Aldrich). Antibody binding was detected using an enhanced chemiluminescence system according to the manufacturer’s protocol (Amersham Biosciences, Piscataway, NJ).