Phospho gskα β

Minimum essential medium-α modification (α-MEM) and fetal bovine serum (FBS) were purchased from Sigma (St. Louis, MO). Collagen gel was obtained from Nitta Gelatin Co. (Osaka, Japan). Bacterial collagenase and dispase were purchased from Calbiochem (San Diego, CA). Antibodies against phospho-Cbl Tyr⁷³⁷, phospho-AKT Thr³⁰⁸, AKT, phospho-ERK, phospho-IKKα/β, phospho-GSKα/β, GSK3β, phospho-AKT substrates and GAPDH were purchased from Cell Signaling Technology (Danvers, MA). Anti-ERK1/2 and anti-p85 antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Cbl, anti-Cbl-b and IKKα antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rhodamine, DAPI and Dead End Fluorometric TUNEL were obtained from Promega (Madison, WI). RANK ligand (RANKL) and MCSF were purchased from R&D Systems (Minneapolis, MN). 1,25-Dihydroxyvitamin D3, prostaglandin E2 and tartrate-resistant acid phosphatase (TRAP) staining kit were obtained from Sigma. FPT III was purchased from Calbiochem and LY294002 from Sigma. Ras activity was determined using a commercial kit purchased from Thermo Scientific (Rockford, IL).

For in vitro studies MLN0128 (Selleckchem Houston, TX, USA) was dissolved in DMSO, phenfomin was dissolved in DMEM. For in vivo studies MLN0128 was dissolved in 1-methyl-2-pyrrolidinone (NMP), then diluted in 15% PEG diluted in water; phenformin was diluted in water at 1.8 mg/ml. Rapamycin was purchased from LC Laboratories (Woburn, MA) and dissolved in DMSO. Antibodies from Cell Signaling Technology (Beverly, MA, USA) used for immunoblots were diluted 1:1,000 and included phospho-p70 S6 kinase (Thr389 #9234), phospho-S6 (Ser235/236 #4858), total 4E-BP1 (#9644), phospho-4E-BP1 (Thr37/46 #2855), phospho-Akt (Ser473 #4060), phospho-Akt (Thr308 #4056), phospho-NDRG1 (Thr346 #5482), Phospho-Tuberin/TSC2 (Thr1462 #3611), phospho-GSKα/β (Ser21/9 #9331), beta-actin (#4970), cleaved PARP (Asp214 #5625), cleaved caspase 3 (Asp175 #9661) and phospho-Raptor (Ser792 #2083). Anti-Hif-1alpha (C-Term #10006421 1:200) antibody was purchased from Cayman Chemical and anti-GLUT1 (GT11-A 1:1,000) antibody was purchased from Alpha Diagnostic International (San Antonio, TX, USA). Plasmid expressing dox-inducible 4EBP1 4Ala (pCW57.1-4EBP1_4xAla, plasmid # 38240) and control vector (pCW57.1, plasmid # 41393) were purchased from Addgene.