Immobilon western blotting detection kit

Cells were harvested by trypsinization, washed with PBS, and lysed in RIPA buffer [25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate-polyacrylamide (SDS) plus a protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan) for 30 min on ice. The lysates were cleared by centrifugation at 16,100 × g for 30 min at 4 °C. Protein concentrations were evaluated using a BCA kit (Thermo Scientific, Rockford, IL, USA). Extracted proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Milford, MA, USA). After blocking with 5% skim milk in TBS-T buffer- for 1 h, the membranes were incubated with primary antibodies for at least 1 h. After washing with TBS-T buffer 3 times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Then the membrane was washed with TBS-T buffer twice and with TBS once. Bands were detected with Chemi-Lumi One (Nacalai Tesque) or the Immobilon Western blotting detection kit (Millipore).

For immunoblotting, HL-60 cells were harvested in 2 days of siRNA transfection. Cells were washed with PBS, and lysed in radioimmune precipitation assay buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxy-cholate, and 0.1% SDS). Lysates were cleared by centrifugation at 16,100×g for 20 min at 4°C. Protein concentration was determined by BCA kit (Thermo Scientific). Extracted proteins were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore). After blocking with 5% skim milk in TBS-T buffer (20 mM Tris at pH 7.5, 150 mM NaCl, and 0.05% Tween 20) for 30 min, the membranes were incubated with anti-TIF-IA (H-300, Santa Cruz) and anti-β-Actin (C4, Santa Cruz) antibodies for overnight at 4°C. After washing with TBS-T buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Bands were detected with Immobilon Western blotting detection kit (Millipore).