Mini protean tetra cell system

Cells (20 × 10⁶) were lysed in RIPA buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM TCEP (Pierce), protease and phosphatase inhibitors (Roche). Lysates were sonicated in a Branson Digital sonicator on ice, centrifuged (4 °C, 16,000 × g for 10 min). Samples were adjusted to 1× LDS sample buffer (life technologies) and 25 mM TCEP was added prior boiling for 10 min. Each lane was loaded with the equivalent of 140,000 CTLs and separated by SDS-PAGE (life technologies NuPAGE precast gels or Bio-Rad Mini-PROTEAN tetra cell system) and transferred to nitrocellulose membranes (Whatman). Blots were probed with the following antibodies: 4EBP1 p-S37/S46 (Cell Signaling Technology (CST) #2855), 4EBP1 - pS65 (CST #9451), 4EBP1 (CST #9452), S6K p-T389 (CST #9239), S6K (CST #9202), Akt p-T308 (CST #4056), Akt p-S473 (CST #4058), SMC1 (Bethyl Laboratories, A300-055A), T-bet (eBioscience 14-5825), IRS2 (CST #4502), PTEN (Santa Cruz sc-7974), FOXO1/3A p-T24/32 (CST #9464), FOXO1 (CST #9454). X-ray films (Konica) were used to monitor chemiluminescence reactions catalysed by HRP-conjugated secondary antibodies. All immunoblots shown are representative of 3 or more biological replicates.