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Figure 5 | BMC Molecular Biology

Figure 5

From: Site-specific acetylation of ISWI by GCN5

Figure 5

Characterisation of an antibody specific forISWIK753ac. (A) Recombinant ISWI was acetylated by GCN5 in the presence of radiolabeled acetyl-CoA. The protein mixture was separated by SDS-PAGE and visualized by Coomassie staining (left panel), by autoradiography (centre panel) and by Western blotting using an antibody raised against an ISWI peptide bearing acetylated K753 (αISWIK753ac). The asterisk points to the position of GCN5 in the gel (lane 2), which autoacetylates itself (lane 4). (B) The antibody does not cross-react with acetylated H3. Recombinant histone H3 was acetylated with GCN5 in presence of radiolabeled acetyl-CoA and analysed as in (A). Western blotting on such a substrate using αISWIK753ac did not reveal any cross-reactivity. (C) ISWI was immunoprecipitated from embryonic nuclear extract using antibodies against ISWI or ISWIK753ac and detected by Western blotting. (D) αISWIK753ac immunoprecipitates were analysed by Western blotting for the presence of acetylated ISWI.

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