Ser473 p akt 1

RPMI-1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Lonza Milano SRL (Milan, Italy). Torin-2, NVP-BGT226, MK-2206 and ZSTK474 were obtained from Selleck Chemicals (Houston, TX, USA). For cell viability determination, Cell Proliferation Kit I (MTT) was purchased from Roche Applied Science (Basel, Switzerland). Annexin V/7-ADD detection kit and cell cycle kits were from Merck-Millipore (Darmstadt, Germany). For Western blot, antibodies to total Akt-1, Ser473 p-Akt-1 and Thr308 p-Akt-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while all the other antibodies were from Cell Signaling Technology (Danvers, MA, USA), including the rabbit secondary antibody. The mouse secondary antibody, z-VAD-fmk, 3-Methyladenine (3- MA), Bafilomycin A1, Ficoll-Paque Plus and phytohemagglutinin were purchased from Sigma Aldrich (Milan, Italy). Dynabeads T-cell separation kit was from Invitrogen life Technologies (Monza MB, Italy). Signals were detected using ECL Plus reagent from Perkin Elmer (Boston, MA, USA).

Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, fetal bovin serum (FBS), nonessential amino acids (NEAA), penicillin and streptomycin were from Lonza (Lonza Milano SRL, Milan, Italy). For cell viability determination, Cell Proliferation Kit I (MTT) was purchased from Roche Applied Science (Basel, Switzerland). Annexin V/7-AAD and cell cycle kits were from Merck-Millipore (Darmstadt, Germany). NVP-BGT226 and Z-VAD-FMK were provided by Selleck Chemicals (Houston, TX, USA). Antibodies to total Akt-1, Ser473 p-Akt-1 and VEGF were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HIF-1α antibody was provided by BD Biosciences Pharmigen, while all the other antibodies were from Cell Signaling Technology (Danvers, MA, USA), including the rabbit secondary antibody, SQSTM1/p62 (#5114) and Cleaved Caspase-7 (Asp198, #9491) antibodies. The mouse secondary antibody, 3-Methyladenine (3-MA), Chloroquine, 1,4-Diazabicyclo[2.2.2.]octane (DABCO) and 4′,6 diamidino-2-pheny-lindole (DAPI) were from Sigma Aldrich (Milan, Italy). Signals were detected with the ECL Plus reagent purchased from Perkin Elmer (Boston, MA, USA).