Figure 4

Immunoprecipitation of H2A.Z-2- and H2A.Z-1-containing mononucleosomes. (A) Top panel: acid-urea-triton (AUT)-PAGE of the histones from HeLa cell mononucleosomes immunoprecipitated with anti-Flag agarose beads. Light chain refers to the immunoglobulin light chain from the anti-Flag agarose beads. Bottom panels: Western blots of anti-Flag, anti-H2A and anti-H4 (loading control) for the histones from immunoprecipitated mononucleosomes separated by sodium dodecyl sulphate (SDS)-PAGE. B.) Two-dimensinal PAGE analysis of HeLa cell histones with the first dimension AUT shown on top and second dimension SDS gel shown below. (C) Western blots of the histones from anti-Flag agarose bead immunoprecipitated mononucleosomes electrophoresed on 15% SDS gels. Samples were normalized with respect to Flag and total H4 levels. Antibodies used to probe the Western blots are indicated on the right. The asterisk indicates the cells were arrested in mitosis by nocodazole treatment prior to immunoprecipitation. The trends in the association of the Flag-tagged proteins with post-translationally modified forms of H3 and H4 were consistent across multiple replicates of the experiment of which a representative example is shown.