Bm chemiluminescence blotting substrate pod

MAF protein levels in both miR-486-3p/NegCTR mimic-transfected and in LMAFvar2IDN/LXIDN-transduced CD34+ cells were detected by western blot analysis. Briefly: cells were harvested, washed twice with ice-cold PBS and lysed (5 × 10⁵ cells/20 μl of lysis buffer) in 50 mM Tris (tris(hydroxymethyl)aminomethane)-Cl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 10 mM KCl, 1 mM EDTA, 20 mM NaF, 0.25% Na doexycholate, 5 mM dithiothreitol (DTT) and protease inhibitors (Complete, catalog #1697498, Roche). Total cellular lysates (15 μg for each sample) were loaded onto 12.5% SDS-polyacrylamide gel and blotted on nitrocellulose membrane. To control loading and transfer, after transfer the membranes were stained by Red Ponceau. Membranes were then preblocked in blocking solution, 5% milk in 0.1% TBST for 1 h at room temperature (RT), incubated with primary antibodies (1 : 2000 dilution of rabbit monoclonal anti-MAF antibody (1 : 2000 dilution, catalog #181188, Abcam, Cambridge, UK; www.abcam.com), at 4 °C overnight of rabbit polyclonal anti–actin antibody (1 : 5000 dilution, Thermo Fisher Scientific Inc., Waltham, MA, USA, catalog #PA1-16889) for 1 h at RT. After three washes with TBST, blots were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1 : 1000 dilution, Thermo Fisher Scientific Inc.) for 1 h at RT and revealed by BM Chemiluminescence Blotting Substrate (POD) (Roche).