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This article contains the following supporting material:
HEp-2 cell lines stably expressing GFP-HP1α (A), GFP-HP1β (B) and GFP-HP1γ (C), and untransfected HEp-2 cells immunostained for endogenous HP1α (D), HP1β (E), and HP1γ (F) were fixed, immunolabeled with anti-centromere autoantibodies and counterstained with the DNA dye ToPro-3. Cells were then analyzed by confocal microscopy. The same confocal section from each channel is shown. Images on the right represent overlays of the GFP-HP1 (green) and the centromere signals (red). Note that all centromere signals are associated with HP1 domains. Larger HP1 domains are always associated with one or several centromeres identifying those structures as pericentromeric heterochromatin. Scale bar: 5 μm.
(A) Schematic representation of the chromatin fractionation procedure. HEp-2 cells were lysed in the presence of 0.3 % NP40 to yield nuclear (N) and cytoplasmic (C) fractions. Nuclei were then digested with 0.2, 1 or 5 units of micrococcal nuclease (MNase) and centrifuged to yield soluble supernatant (S) and insoluble pellet (P) fractions. Soluble supernatant fractions containing mononucleosomes were used for co-immunoprecipitation assays (IP, see Fig. 1). (B) DNA extracted from equivalent S and P fractions after MNase digest were electrophoresed in a 1.5 % agarose gel followed by ethidium bromide staining. The migration position of mononucleosomes is indicated (mononucleos.). (C) Whole cell protein extracts (WCE) and aliquots of the chromatin fractionation procedure from HEp-2 cells (blots designated acK-H3, meK9-H3, CENP-A, HP1α, HP1β, and HP1γ) and GFP-HP1 expressing HEp-2 cells (blots designated GFP-HP1α, GFP-HP1β, and GFP-HP1γ) were subjected to 17.5 % SDS-PAGE and western blotting using antibodies against N-terminally acetylated histone H3 (acK-H3), histone H3 di-methylated at lysine 9 (meK9-H3), centromere protein A (CENP-A), HP1α, HP1β, HP1γ, and anti-GFP antibody (GFP-HP1α, GFP-HP1β, and GFP-HP1γ). Nucleosomes containing acK9-H3 or meK9-H3 are released from pellet fractions into soluble supernatant fractions with increasing amounts of MNase. A similar behavior is observed for endogenous as well as GFP-tagged HP1 proteins indicating similar biochemical properties of these proteins during MNase fractionation. In contrast, CENP-A-containing nucleosomes are not released into soluble fractions under these conditions (CENP-A).
Prepared by: the Journals Production Manager
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