Figure 3. Biochemical purification of Beaf32 complex identifies CP190 as a major co-factor.

A. Affinity chromatography procedure to purify the Beaf32 complex followed by competition with either the C-terminal peptide of Beaf32 (panel B) or peptides covering the entire Beaf32 sequence (panel C).

B. Coomassie-stained SDS-PAGE gel after elution of affinity-purified complex with the C-terminal peptide as a competitor added on beads coupled to purified anti-Beaf32 antibodies or IgG control (see Figure S2A; Extended Experimental procedures). The arrow shows the CP190 protein as confirmed by mass-spectrometric analysis (144 peptides versus 2 peptides in IgG control)(see also Supplementary Figure S2B–C).

C. Following affinity chromatography, Beaf32-CP190 interactions were competed by adding one of the (18AA-long) peptides spanning the entire Beaf32 (see Experimental procedures). The elution by addition of each peptide was assessed by western blotting using anti-CP190 (see Figure S2B) or anti-histone H3 antibodies as loading control. The sequence of peptide number 10 (‘p10’) is SEDPLCYSPIHVMDDEGL. ‘100x’ indicates the exposure time (approx. 30 min) to detect H3. (See also Supplementary Figure S3).

D. Two-hybrid assays probing the interactions between wild-type or mutant Beaf32 and CP190. For Beaf32 mutants, 2 or 4 mutations (‘Beaf32-M1/2’, respectively; see Experimental procedures) were introduced into the region corresponding to ‘p10’ (see panel C). SD+/−ura corresponds to the same batch of cells spot onto media containing uracyl (growth control) or not, respectively.