Cst2947

Total protein was extracted from indicated cells or tumor tissues using lysis buffer (2% SDS, 100 mM DTT, 10 mM Tris (pH 6.8) and 10% glycerol). Tumor tissues in lysis buffer were sonicated at a frequency of 70 Hz for 180 s. Then they were centrifuged at 12000 rpm for 5 min and boiled at 98°C for 10 min. The protein was separated on a 12% or 15% SDS-PAGE gel and transferred to polyvinylidenefluoride membrane (PVDF; Millipore, U.S.A.). The membranes were blocked with 5% silk milk at room temperature for 1 h and incubated with primary antibodies at 4°C overnight. Antibody against ENO1 (ab85086, 1:1000), cleaved caspase 3 (ab2303) and 9 (ab2324) was from Abcam (U.K.). Antibodies against cyclin D1 (sc-8396, 1:500), E1 (sc-377100, 1:500), p21 (cst2947, 1:1000), p27 (cst3686, 1:1000), caspase 3 (cst9662, 1:1000) and 9 (cst9502, 1:1000) were from Cell Signaling (U.S.A.) and Santa Cruz Biotech (U.S.A.). Antibody against β-catenin (cst8480, 1:1000) was from Cell Signaling (U.S.A.). GAPDH antibody was from ABclonal (AC033, 1:3000) (U.S.A.). All the secondary antibodies (sc-2537 and sc-2005, 1:5000) were from Santa Cruz Biotech (U.S.A.).

Cells were homogenized with a Tris-SDS-EDTA Lysis buffer containing protease and phosphatase inhibitors and beta-mercaptoethanol. Samples were boiled and run on SDS-PAGE for western blotting according to standard procedures. Nitrocellulose membranes were probed for p21 (1:1000, CST 2947, Cell Signaling Technology), p16 (1:1000, CST 80772, Cell Signaling Technology), β-Actin (1:200, sc-81178, Santa Cruz Biotechnology), Vinculin (1:200, sc-73614, Santa Cruz Biotechnology), γH2A.X (1:200, sc-517348, Santa Cruz Biotechnology), H2A.X (1:200, sc-517336, Santa Cruz Biotechnology), pAkt (1:1000, CST 9018, Cell Signaling Technology), PP2A-A (1:1000, CST-9780, Cell Signaling Technology), Akt (1:1000, CST 2938, Cell Signaling Technology), and Phospho-Akt1 (Ser473) (D7F10) (1:1000, CST-9018, Cell Signaling Technology). Blots were detected with the Azure Biosystems ECL detection kit (AC2103). All gels derived from the same experiment and were processed in parallel (Supplementary Fig. 13). Quantification of band intensities by densitometry was carried out using ImageJ.