Western blot analysis of Akt, p-Akt (Ser473), GSKβ and p-GSKβ (ser-9) protein expression

Protein extracts and kinases expression was determined as described previously¹⁰. MDA-MB 468 cells were treated with the PI3K/Akt/mTOR inhibitors alone or in combination with GSK3 inhibitor SB216763 for 24 h and then washed with 5 ml ice cold PBS and the cells harvested by scraping with 150 μl of lysis buffer (PBS containing 1 mM DTT, a protease inhibitor cocktail, 10 mM sodium pyrophosphate and 1 mM sodium orthovanadate). Samples were kept frozen until they were lysed with a probe sonicator for 30 secs. Protein concentration quantification was carried out with the bicinchonininic acid (BCA) assay. Lysates (25 μg protein/well) were resolved on 4–12% acrylamide Bis–Tris gels (Invitrogen) and the proteins transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, UK) at 30 V for 1.5 h using a wet transfer system (Invitrogen, UK). Membranes were blocked for 1 h at room temperature in PBS containing 0.1% Tween 20 (PBST), and either 50% sea block for Akt and GSKβ antibodies or 1% (w/v) BSA for p-Akt and p-GSKβ antibodies. Membranes were incubated overnight at 4 °C in PBST containing the appropriate blocking buffer and antibodies at either 1:667 dilution (Akt and p-Akt) or 1:1000 dilution (GSK and pGSK). Membranes were washed four times in PBST prior to 1 h incubation with the appropriate anti-rabbit fluorescent secondary antibody (Licor) in PBST in the dark at (1/10000 dilution). Blots were drained and wrapped in cling film to prevent drying. Blots were imaged using the Odyssey® CLx LI-CORE imaging system to detect fluorescence at 800 nM. Images were then analysed using the Odyssey Image Studio Lite (LI-CORE) software. Each protein ran at the expected molecular weight as previously observed for the Akt and GSK3 proteins. There was only one band on each blot and both the phosphor- and non-phospho version of each protein ran at exactly the same molecular weight. The Akt and pAkt antibodies were used and characterised by our group previously¹⁰. Full blots for GSK3 and pGSK3 are shown in Supplementary Fig 1 and for Akt and pAKT are shown in Supplementary Fig 2.