Impact of signal transduction inhibitors on Akt signalling. (a) The MDA-MB-453 cells were treated for 2 hours with Ly294002 (30 μmol/l), OSU03012, OSU03013 (5 or 10 μmol/l), or celecoxib (50 or 75 μmol/l). The Celecoxib analogues inhibited Akt phosphorylation at both threonine 308 and serine 473. Phosphorylation of the Akt substrates glycogen synthase kinase (GSK) and 4E binding protein (4EBP)-1 was subsequently attenuated. In contrast, celecoxib did not have an inhibitory effect. Ly294002 inhibited signal transduction through Akt, as expected. Total Akt and actin were unaffected by exposure to the signalling inhibitors. (b) T47D cells were also exposed to the drugs as described above and evaluated for phosphorylated Akt (P-Akt) using antibodies to Ser473 and Thr308. Total Akt was included as a control for loading. (c) Cells were treated as above and Akt kinase was measured against the substrate GSK. Each of the celecoxib analogues inhibited Akt kinase activity. The degree of inhibition was similar to that with Ly294002. There was minimal nonspecific kinase activity in the absence of Akt based on the IgG control. Total Akt was evaluated as a loading control to confirm that the loss of activity was not due to differences in experimental conditions. (d) The MDA-MB-453 cells were treated for 2, 4, or 6 hours with dimethyl sulphoxide (DMSO), Ly294002 (30 μmol/l), OSU03012 (10 μmol/l), or OSU03013 (10 μmol/l) and probed for P-Erk1/2, total Erk, P-MK2, total MK2 and actin.