Anti acetylated histone 3

Liver (1 g) was crosslinked with 1% formaldehyde for 7 min at room temperature before stop by adding 0.5 M glycine. After sonication, fragmented chromatin was incubated overnight at 4 °C with anti-ZBTB20 antibody 9A10 (home-made, 1 μg per reaction), anti-acetylated histone 3 (Millipore, Cat #17-10050, 0.5 μg per reaction) as positive control or isotype IgG (Upstate, 1 μg per reaction) as negative control, and followed by incubation with Dynabead-conjugated Protein G (Invitrogen). Purified chromatin DNA was subjected to real-time PCR analysis with the primers for gene promoter, the sequence of which is were listed in Supplementary Table 5. The data were analysed using the formula of 2^−ΔΔCt, where ΔΔCT=(Ct[IP]−Ct[input])_SA−(Ct[IP]–Ct[input])_NS. SA=specific antibody, NS=non-specific antibody. Three independent ChIPs were performed.

Western blot analysis was performed as described previously described (Oehme et al. 2009b (link)). The following antibodies were used: anti-histone 3 (#9715, Cell Signaling Technology, Leiden, The Netherlands), anti-acetylated histone 3 (#06-911, Millipore), anti-tubulin (#2148, Cell Signaling Technology), anti-acetylated tubulin (#6793, Sigma-Aldrich), anti-acetylated SMC3 (kindly provided by Katsuhiko Shirahige, Institute for Molecular and Cellular Biosciences, University of Tokyo, Japan (Nishiyama et al. 2010 (link))), anti-HSC70/HSP70 (#sc-33575, Santa Cruz Biotechnology, Heidelberg, Germany), and anti-β-actin (#5441, Sigma-Aldrich).

Fragmented chromatin from GH3 or MMQ cells was incubated overnight with anti-ZBTB20 antibody 9A10 (home-made, 0.5 μg per 10⁶ cells), anti-acetylated histone 3 (Millipore, 0.5 μg per 10⁶ cells) as positive control, or isotype IgG (Upstate, 0.5 μg per 10⁶ cells) as negative control, and followed by incubation with Dynabead-conjugated Protein G (Invitrogen). Purified chromatin DNA was subjected to real-time PCR analysis with the primers for rat Prl promoter, the sequence of which is 5′-TTTGGGGTCAGAAGAGGC-3′ and 5′-TTGTGGAAGGAGCGCAGT-3′. The data were analysed using the formula of 2^–ΔΔCt, where ΔΔCT=(Ct[IP]–Ct[input])_SA– (Ct[IP]–Ct[input])_NS. SA=Specific antibody, NS=Non-specific antibody. Three independent ChIPs were performed.