Download Methylation of histone H3-lysine 27 by EED-EZH2/ESC-E(Z)

Wang et al.
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Role of H2A Ubiquitination in Polycomb Silencing
Hengbin Wang, Liangjun Wang, Hediye Erdjument-Bromage,
Miguel Vidal, Paul Tempst, Richard S. Jones and Yi Zhang
Supplementary Material
Supplementary data
Figure S1. The monoclonal uH2A antibody recognizes uH2A from Drosophila SL2 cells. a,
Sequence alignment of human and Drosophila H2A around the ubiquitination site. b, Western
blotting of histone extracts from SL2 cells using a well-characterized monoclonal antibody
(E6C5) against uH2A 1. c, Coomassie staining of a parallel gel containing the same sample as in
b and mass spectrometric analysis of the indicated band. The identified peptides confirm that the
protein is ubiquitinated H2A. In addition to the specificity test shown here, the antibody was
previously subjected to extensive tests in the original paper1 that included: 1) detection of a
single band on a 1-D western blot of crude protein lysate from WI-38 cells; 2) detection of a
Wang et al.
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single spot on a 2-D western of total nuclear pellet proteins from AMA cells and identification of
both H2A and ubiquitin peptides in the spot by mass spectrometry analysis; 3) demonstration
that the antibody is not able to recognize 2 g of purified ubiquitin on a 1-D western.
1.
Vassilev, A. P., Rasmussen, H. H., Christensen, E. I., Nielsen, S. & Celis, J. E. The levels
of ubiquitinated histone H2A are highly upregulated in transformed human cells: partial
colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase. J
Cell Sci 108 ( Pt 3), 1205-15 (1995).