Akt activity assay Akt activity was assayed using a non radioactive assay equipment purchased from Cell Signaling Technology. Samples were centrifuged and supernatants were assayed for protein content. Aliquots containing equal amount of protein were added to agarosFingolimod distributor e cross linked to mouse monoclonal anti Akt antibody and incubated over night at 4 C with continuous rocking. The beans were then cleaned with cell lysis buffer and with kinase assay buffer containing 25 mM Tris, 5 mM w glycerophosphate, 2 mM dithiothreitol, 0. 1 mM sodium orthovanadate and 10 mM MgCl2. Thereafter, the beads were re-suspended in kinase assay buffer supplemented with 0. 20 mg and 2 mM ATP mL 1 of glycogen kinase synthase 3a/b crosstide and the samples were incubated for 30 min at 30 C. The reaction was stopped by the addition of sample buffer, the samples were heated at 100 C and analysed by Western blot utilizing a rabbit polyclonal antibody against phospho Ser21/9 GSK 3a/b. Three separate cell preparations were examined. Statistical analysis Results are reported as mean SEM. Kinetic data and concentration response curves were analysed by non-linear regressioPlastid n curve fitting using the system Graph Pad Prism. Statistical analysis was performed by either Students unpaired t test or one way ANOVA followed by Newman Keuls post hoc test as appropriate. Materials 2 deoxy D glucose and 3 OMG were obtained from Analytical Sciences and PerkinElmer Life. Cell tradition materials including Hams F12 medium, FCS, penicillin streptomycin and hygromycin were obtained from Invitrogen. DPDPE, naltrindole, naloxone, 3 OMG, dibutyryl cyclic AMP, phorbol 12 myristate 13 acetate, mouse recombinant insulinlike growth factor, pertussis toxin, wortmannin, tyrphostin I OMe AG 538, phloretin, cytochalasin B, phosphatase inhibitor cocktail 1, okadaic acid, protease inhibitor cocktail and streptavidin conBortezomib molecular weight jugated agarose were from Sigma Life Science. 2 Deoxy D sugar, Go 6850, Go 6983, PP2, PP3, Akt inhibitor VIII, phosphatidylinositol 3 kinase an inhibitor VIII PI3 Kg inhibitor II and myristoylated PKCz pseudosubstrate inhibitor, tyrphostin AG 1024 and tyrphostin AG 1478 were from Calbiochem. SNC 80, LY294002, LY303511, PD 98059 and U0126 were from Tocris Cookson Ltd. Sp cAMPS was from Biomol GmbH. The key antibodies used were from these sources: rabbit polyclonal anti GLUT1 from Millipore, mouse monoclonal anti GLUT3, mouse monoclonal anti Na /K ATPase a1 subunit, rabbit polyclonal anti Akt1/2/3 and anti PKCz from Santa Cruz Biotechnology, rabbit polyclonal antiphospho Thr308 Akt, rabbit polyclonal anti PI3K p110a, PI3K p110b, PI3K p110g, p44/42 MAP, phospho Tyr416 Src, phospho Thr410/ 403 PKCz/l, rabbit monoclonal anti Src and anti phospho Thr308 Akt from Cell Signaling Technology, rabbit polyclonal to dually phosphorylated ERK1/2 from Neuromics, and rabbit polyclonal anti GLUT4 and actin from Sigma.