Immobilon western detection reagent

Western blots were performed as described previously32 (link). Cells or tissue lysates were incubated with RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM NaF, and 5 mM Na₃VO₄) containing protease inhibitors. Proteins were separated by 8~12% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were probed with the indicated antibodies and were developed using Immobilon Western Detection Reagents (Millipore, Billerica, MA, USA).

Cells were lysed in RIPA lysis buffer [150 mmol/L NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 50 mmol/L Tris‐HCl pH 7.5, 2 mmol/L EDTA, 1 mmol/L PMSF, 1 mmol/L DTT, 1 mmol/L Na₃VO₄, and 5 mmol/L NaF] with protease inhibitor cocktail and sonicated. Approximately 30 μg of protein was separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk for 1 hour, proteins were incubated with the primary antibodies overnight: anti‐cyclin D1 (1:1000), anti‐HDAC1 (1:1000), anti‐HDAC2 (1:1000), anti‐HDAC4 (1:1000) and anti‐HDAC5 (1:1000). As secondary antibodies, horse radish peroxidase‐linked antimouse or anti‐rabbit antibodies were used. Protein bands were visualized using Immobilon Western Detection Reagents (Millipore, Billerica, MA, USA). The Bio‐ID software was used to quantify protein expression (Vilber Lourmat, Eberhardzell, Germany).

The total cellular proteins were extracted with a lysis buffer (20 mMTris-HCl, pH 8.0, 50 mMNaCl, 1% Nonidet P40 (Sigma), 1% Triton X100), 1% (v/v) phenylmethanesulfonyl fluoride (PMSF, Sigma), and 1% (v/v) protease inhibitor cocktail (nacalaitesque) (PI; pH 7.4). The cell lysates were centrifuged at 15,000g for 15 min at 4°C to achieve protein sample solutions. Proteins with sample buffer were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on polyacrylamide gels (7.5% and 10%) and electrophoretically transferred onto a polyvinylidenedifluoride (PVDF; Immobilon- P, Millipore) membrane. The membranes were incubated with primary antibodies of mouse anti-β-actin (1: 2000; Sigma-Aldrich), anti-pAkt (1: 1000; Cell Signaling), total-Akt (Cell Signaling) and β-catenin (1: 2000; BD Bioscience) for 2 h at room temperature. The incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000 dilution; Jackson ImmunoResearch Laboratories) was performed for 2 h at room temperature. HRP activity was assayed with the Immobilon Western detection reagents (Millipore) according to the manufacturer’s instruction.

Western blots were performed as described previously 16. Cells lysates were prepared in RIPA buffer (150 mM NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 50 mM Tris‐HCl, pH 7.5, 2 mM EDTA, 1 mM PMSF, 1 mM DTT, 1 mM Na₃VO₄, 5 mM NaF) containing protease inhibitors. Proteins were separated by 8% SDS‐PAGE and were then transferred to polyvinylidene difluoride membranes. The membranes were probed with the indicated antibodies and developed using Immobilon Western Detection Reagents (Millipore, Billerica, MA, USA). Protein expression was quantified using Bio‐ID software (Vilber Lourmat, Germany).