Histone 2b

Western blotting was performed as described previously [7 (link)]. The protein levels of Acetyl-Histone H3, Histone H3, Histone 2B, Bcl-2, and cleaved caspase-3 were analyzed with the following antibodies, respectively: Acetyl-Histone H3 (Millipore, Temecula, CA), Histone H3 (Millipore, Temecula, CA), Histone 2B (Santa Cruz, Santa Cruz, CA), Bcl-2 (Cell Signaling, Beverly, MA), and cleaved caspase-3 (Asp175) Rabbit pAb (Cell Signaling, Beverly, MA). The membranes were developed on a Molecular Imager ChemiDoc XRS+ System (Bio-Rad, Hercules, CA) and the intensity of the protein band was analyzed by ImageJ software (1.46b, National Institutes of Health, USA). All Western blot analyses were performed in triplicate.

Cells from control and ethanol treated groups were collected and the nuclear fractions of NCCs were generated using the TransFactor extraction kit (Clontech, Mountain View, CA) following the manufacturer's protocol. Briefly, cells were rinsed twice with ice-cold PBS before they were scraped in ice-cold PBS. Cells were resuspended in lysis buffer, incubated for 15 min., and centrifuged for 5 min. at 450 X g. The cell pellet was resuspended in lysis buffer, homogenized with 15 strokes of a homogenizer, and centrifuged at 10,000 X g for 20 min. Then the crude nuclear pellet was resuspended in buffer and disrupted with 15 strokes of a homogenizer. The nuclear suspension was shaken gently for 30 min at 4°C and the supernatant was collected by centrifuging at 20,000 X g for 5 min. This fraction is the nuclear extract. The protein concentrations were determined using Pierce® BCA protein assay. Fraction purity was assessed using nuclear marker histone 2B and cytosolic marker IkB-α (Santa Cruz, Santa Cruz, CA). Siah1 nuclear accumulation was analyzed by determining the Siah1 protein levels in nuclear fractions using Western blot.