Hybond c extra nitrocellulose

BCSCs were collected, washed twice in ice-cold PBS and then resuspended in ice-cold buffer containing TPER Reagent (Pierce) 300 mM NaCl (Sigma Aldrich) 1 mM orthovanadate (Sigma Aldrich) 2 mM pefabloc (Roche), proteinase inhibitor cocktail (aprotinin, pepstatin and leupeptin, each at 5 μg/mL, Sigma Aldrich) and incubated for 30 min on ice. Protein extracts were resolved using a SDS-PAGE gel and separated by electrophoresis, transferred to nitrocellulose membranes (Hybond-C Extra, Nitrocellulose, Amersham) and subsequently blocked with PBS Tween 20 0.1% (Sigma Aldrich) not-fat dry milk 5% (Sigma-Aldrich) for 1 hour at R.T. Immunodetection was performed by incubating the membranes with the primary antibodies at 4°C O.N., and finally with secondary antibodies for 1 hour at R.T. The following antibodies were used: p63-α (D2K8X, rabbit IgG, CST), p63 (4A4, mouse IgG2A, Santacruz), p110-α (PI3K, C73F8, rabbit IgG, CST), AKT (rabbit, CST #9272), P-AKT (D9E, rabbit, IgG, CST), β-ACTIN (8H10D10, mouse IgG_2b, CST), anti-mouse HRP-conjugated (goat IgG, ThermoFisher Scientific), anti-rabbit HRP-conjugated (goat IgG, ThermoFisher Scientific). Immunoreactive bands were detected using SuperSignal™ West Dura Extended Duration Substrate (ThermoFisher Scientific) and revealed by using the Amersham imager 600 (GE Healthcare).

Cellular pellets were lysed in RIPA buffer: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA and 1% Triton-X100 and protease inhibitors (Sigma-Aldrich Inc., Saint Louis, MO). Samples were resolved on 4–12% SDS-PAGE gels using a mini-gel apparatus (Bio-Rad Laboratories, Richmond, CA) and transferred to Hybond-C extra nitrocellulose (Amersham Pharmacia Biotech, Piscataway, NJ). Membrane was blocked for 1 h with 5% non-fat dry milk in TBS containing 0.05% Tween-20 and incubated over night with primary antibody. The primary antibodies used are: pAKT S473 (4058) and pERK T202/Y204 (9101) (all from Cell Signaling Technology, Danvers, MA) and GAPDH (G9545) (Sigma-Aldrich Inc., Saint Louis, MO). Washed filters were then incubated for 45 min with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) and visualized by using an enhanced chemioluminescence detection system (ChemiDoc XRS+ (Bio-rad) with Image Lab software).