Histone 3

Western blot analysis was performed using standard techniques. In brief, differentiating CD34⁺ progenitors were lysed in Laemmli buffer [0.12 mol/L Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.05 μg/μL bromophenol blue, and 35 mmol/L β-mercaptoethanol], sonicated, and boiled for 5 minutes. Equal amounts of total lysate were analyzed by 12% sodium dodecylsulfate polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), incubated with blocking buffer (Tris buffered saline/Tween 20) containing 5% low-fat milk for 1 hour at room temperature before incubation with antibodies against acetyl-histone 3 (lysine 27) (Millipore, Billerica, MA) or histone 3 (Millipore, Billerica, MA) overnight at 4°C in a buffer containing Tris buffered saline/Tween 20 with 5% bovine serum albumin (BSA) (Sigma-Aldrich, Zwijndrecht, the Netherlands). Blots were subsequently incubated with peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) for 1 hour at room temperature. Chemiluminescence was used as a detection method according to the protocol of the manufacturer (Odyssey, Amersham Pharmacia, Amersham, UK).