P akt sc 7985 r

Immunoblotting and co-immunoprecipitations were performed as described [48 (link)]. Antiserum against FOXO (C29H4) and anti-Flag M2 (F3165) was purchased from Cell signaling Technology and Sigma for immunoblot analysis. E2F1 antibody (C20) was purchased from Sigma. Anti-HA antibody was purchased from Roche (3 F10). Anti-p53 (sc-6243, Santa Cruz), p-FOXO1 (9461S, Cell Signaling Technology), p-Akt (sc-7985-R, Santa Cruz), Capase-3 (Cell Signaling Technology, Cat# 9661), Ki-67 (SP6) (Biocare Medical, Cat# CRM325), and TUNEL (EMD Millipore, Cat# S7101) were used for IHC. IHC detection was through primary antibodies listed in Additional file 1: Table S2, with Vector biotinylated secondary (1:250), tertiary was streptavidin-horseradish peroxidase (Covance #SIG-32000) and chromagen 3,3′-diaminobenzidine substrate (Covance #SIG-31043).

Heat-inactivated fetal bovine serum (FBS), trypsin / EDTA and Dulbecco’s modified Eagle’s medium (DMEM) containing penicillin (100 UI/mL) and streptomycin (100 μg/mL) were from Gibco (Grand Island, NY) or Cultilab (Campinas, SP). Unless stated otherwise, other reagents and chemicals used were from Sigma-Aldrich (St. Louis, MO). P-Akt (sc-7985-R), p-Erk (sc-7383) and Erk 1/2 (sc-135900) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Akt (#9272), p21 (2946S), PARP (#9542), cleaved caspase-3 (#9661) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB- 166, American Type Culture Collection; Manassas, VA). Mouse anti-β-actin was purchased from Sigma-Aldrich.