Anti acetylated histone h4 k16

Chemical treatments of embryos from dmd+/− crosses were performed as above. At 4 dpf, heads were cut from anesthetized animals and were arrayed in 96-well plates for genotyping. The remaining trunk and tail portions of the animals were arrayed in separate 96-well plates, to which LDS sample buffer (Invitrogen NP0007) was added and stored at −20⁰C until genotyping was completed. Trunk lysates were then pooled by genotype. Approximately, 0.75 animal equivalent per lane was separated on 12% NuPAGE Bis-Tris gels run in NuPAGE MES buffer (Invitrogen) and blotted. Blots were probed separately with anti-pan acetylated histone H4 (1:2000, rabbit polyclonal, Millipore Cat. # 06-866) and anti-acetylated histone H4 K16 (1:2500, rabbit polyclonal, Millipore Cat. # 07-329). Anti-actin was included with both anti-acetylated histone antibodies as a loading control (1:2000; mouse monoclonal Clone C4, MP Biomedical Cat. # 69100). Infrared-dye labeled secondary antibodies (Rockland) were visualized using a Licor Odyssey infrared scanner.

Cells were harvested by trypsinization and acid extraction of histone proteins was carried out by lysing cells in PBS with 0.5% Triton X-100, 2 mM phenylmethyl sulphonyl fluoride (PMSF) and 0.02% NaN₃. Nuclei were pelleted by centrifugation at 1,000g and the nuclear pellet was incubated at 4 °C overnight in 0.2 N HCl 63 (link). Western blot analysis was carried out as described above using 5 μg of acid-soluble lysate per sample. Antibodies used were as follows: anti-histone H3 (Cell Signaling #9715, 1:1,000), anti-acetyl-histone H3K9 (Cell Signaling #9649P, 1:1,000), anti-histone H4 (Millipore 07-108, 1:250) and anti-acetylated histone H4K16 (Millipore 07-108, 1:500).