Anti acetyl histone h4 lys16

Proteins extracted from treated cells were transferred to polyvinylidene difluoride membranes and blotted with rabbit anti-β-tubulin (1:4000) (Santa Cruz Biotechnology), anti-trimethyl-histone-H3 (Lys27) (1:1000) (Millipore), anti-acetyl-histone H4 (Lys16) (1:1000) (Millipore), mouse anti-histone H3 (1:1000) (Active Motif, CA), MLH1 (BD Biosciences) and actin (Abcam). Goat anti-Rabbit IgG (H+L), peroxidase labeled antibodies (1:4000) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) were used for detection of H4K16ac and H3K27me3. Infrared-dye-labeled anti-mouse antibodies (Licor Bioscience) and the Odyssey infrared imaging system (Licor Biotechnology) were used to detect MLH1.
Densitometry used Image J analysis software. Experiments were done in triplicate.

The expression plasmid constructs containing p49/STRAP and empty vector were transfected into C2C12 cells using Lipofectamine 2000 (Invitrogen) as previously described (Zhang et al., 2004 (link)). At 24 h after the transfection, cells were harvested, and the whole-cell lysate was isolated. The immunoprecipitation and Western blotting were carried out as previously described (Zhang et al., 2004 (link)). Antibodies that were employed included p49/STRAP antibody (Zhang et al., 2004 (link)), Histone H4 (Millipore), anti-acetyl-Histone H4 (Lys16) (Millipore), MFN1 and MFN2 (Santa Cruz Biotechnology) antibodies. Secondary antibodies are from LI-COR Inc. The Western bots were imaged and the bands were analyzed using Odyssey Imaging Systems (LI-COR Inc.).