Western blotting

K562 cells were treated with DMSO, S1P (0.5, 1, 1.5 or 2 µM for 8 h or 1 µM for 2, 4, 8, 12 or 24 h), DMS (5, 10 or 20 µM for 24 h), EX527 (5, 10, 20 or 40 µM for 24 h), SKI-II (20 µM for 24 h) or SKI-II + EX527 (20 µM EX527 and 20 µM SKI-II for 24 h). TF-1-T315I cells were treated with DMSO, EX527 (5, 10, 20 or 40 µM for 24 h), SKI-II (20 µM for 24 h) or SKI-II + EX527 (20 µM EX527 and 20 µM SKI-II for 24 h). Following this, the cells were washed twice with 1X PBS and proteins were extracted from cells by suspension in RIPA buffer (Sigma-Aldrich; Merck KGaA) containing PMSF (Sigma-Aldrich; Merck KGaA) and 1% phosphatase inhibitors (Sigma-Aldrich; Merck KGaA). Lysates were clarified by centrifugation at 12,000 x g, 4˚C for 30 min and total protein was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Then, 30 µg of sample protein was separated by 12% SDS-PAGE and transferred to PVDF membranes. Following blocking with 5% non-fat dry milk in TBS with 0.05% Tween-20 at room temperature for 2 h. The membranes were incubated with primary antibodies against SIRT1 (cat. no. 2496; 1:1,000 dilution; Cell Signaling Technology, Inc.), ERK (cat. no. 4695; 1:1,000 dilution; Cell Signaling Technology, Inc.), phosphorylated (p-)ERK (cat. no. 9101; 1:1,000 dilution; Cell Signaling Technology, Inc.), STAT5 (cat. no. 25656; 1:1,000 dilution; Cell Signaling Technology, Inc.), p-STAT5 (cat. no. 9359; 1:1,000 dilution; Cell Signaling Technology, Inc.), β-actin (cat. no. 4970; 1:1,000 dilution; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 2118; 1:1,000 dilution; Cell Signaling Technology, Inc.). Subsequently, the membranes were incubated with an anti-rabbit secondary antibody conjugated with peroxidase (cat. no. ZB-5301; 1:5,000 dilution; OriGene Technologies, Inc.). Signals were detected using a chemiluminescent detection system (Pierce; Thermo Fisher Scientific, Inc.).