Ac h4

Western immunoblotting was conducted with 4T1.2 cells. Cells were treated with 0.1% DMSO (control) or indicated concentrations (1 μM or 5 μM) of SAHA or POEG-b-PSAHA, respectively. After 24 h treatment, cells were rinsed twice with ice-cold PBS and lysed by RIPA buffer. The lysates were centrifuged at 12,500 rpm for 10 min. Samples with equal amounts of total cellular proteins were subjected to sodium dodecylsulfate polyacryl amide gel electrophoresis (SDS-PAGE), followed by transferring to nitrocellulose membranes. The membranes were first blocked in 5% nonfat dry milk dissolved in DPBS with 0.1% Tween 20 (PBST) for 1 h at RT, and then incubated with primary antibody at a final dilution of 1:1,000 in 5% BSA in PBST overnight at 4 °C. After washing three times with PBST, the membranes were incubated with secondary antibody at a final dilution of 1:5,000 in PBST for 1 h at room temperature. After washing three times with PBST, bound antibodies were detected by chemiluminescence. Beta-actin was used as the loading control. Primary antibodies for AC- H3 and AC-H4 were from Cell Signaling Technology (MA, USA) and the antibody for beta-actin was from Sigma-Aldrich (MO, USA).