Gel running system

Cells were harvested and lysed in TNE lysis buffer (20 mM Tris-HCl [pH 7.5], 1% NP-40, 150 mM NaCl, 2 mM EDTA [pH 8.0], 50 mM NaF, 1 mM Na₃VO₄) supplemented with the appropriate protease inhibitors. For tissue samples, the dissected tissue was homogenized in pre-chilled RIPA buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na₃VO₄, 1 mM NaF) with protease inhibitors using a tissue grinder. Total protein was quantified by the Bradford assay. Samples containing equal amounts of total protein were denatured by boiling for 5 min in 4× sodium dodecyl sulfate (SDS) sample buffer. To prepare phospho-proteins, 1× SDS sample buffer was directly applied to the cells. The cells were harvested, sonicated, and boiled at 98 °C for 5 min. SDS-polyacrylamide gel electrophoresis (PAGE) was performed using a Bio-Rad gel running system (Bio-rad, Hercules, CA, USA). Separated proteins on the gel were transferred to polyvinylidene difluoride (PVDF) membranes. For blocking, 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) was used. Membranes were incubated with specific primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. After three washes with TBST for 5 min, the immunoblots were visualised using an enhanced chemiluminescence (ECL) system.

The first dimension, isoelectric focusing (IEF) electrophoresis, was performed using 18 cm, pH 3–10 non-linear IPG strips (Bio-Rad, Hercules, CA, USA). Each sample was diluted in rehydration buffer, which contained 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.5% ampholytes at pH 3–10 and Bromophenol blue to a final volume of 340 mL containing 250 μg protein. The IPG strip was rehydrated for 14 h at room temperature. Isoelectric focusing with Protean IEF Cell (Bio-Rad, Hercules, CA, USA) was performed at 20 °C using the following protocol: 1000 V for 2 h, 4000 V for 1 h, 8000 V for 1.5 h, and keep 8000 V for 9 h. After IEF, the IPG strip was equilibrated in two equilibration solutions for 15 min each with gentle shaking. The first equilibration solution contained 6 M urea, 2% SDS, 20% glycerol, 0.05 M Tris-HCl (pH 8.8), and 2% DTT. In the second equilibration solution, DTT was replaced by 2.5% iodoacetamide. For the second dimension, SDS-PAGE, the IPG strip was transferred to a homogeneous polyacrylamide gel (12%, 200 × 230 × 1.0 mm) and the electrophoresis was performed using a gel running system (Bio-Rad, Hercules, CA, USA) at a current of 40 mA per gel for 4 h (see Figure S2).