Skip to main content
. 2000 Oct;20(20):7572–7582. doi: 10.1128/mcb.20.20.7572-7582.2000

FIG. 5.

FIG. 5

Ikaros, NuRD, and SWI/SNF proteins coimmunopurify from chromatography fractions containing peak PYR complex DNA-binding activity. (A) Ikaros and Mi-2 coimmunoprecipitate from reactive yellow 3-agarose-fractionated MEL extract. (Left panel) Western blot analysis with rabbit polyclonal anti-Ikaros antibody. Mouse monoclonal antibodies used for immunoprecipitation (IP) are indicated above lanes 2 through 4. The sample for lane 1 was 1/10 the volume of reactive yellow 3-agarose-purified PYR complex (yellow 3 fraction 67 [Y3]) before precipitation as a positive control for the Ikaros signal (Ik-1, 65 kDa; Ik-2, 55 kDa). Weak cross-reaction of the anti-rabbit IgG secondary antibody with the mouse IgG heavy chain results in a faint band of approximately 50 kDa in lanes 2 through 4. (Right panel) Western blot analysis with an anti-Mi-2 rabbit polyclonal antibody. Lane 1, reactive yellow 3-agarose fraction 67; lane 2, anti-Aiolos precipitate; lane 3, anti-Mi-2 precipitate; lane 4, anti-Ikaros precipitate. (B) Ikaros, BRG1, and Mi-2 copurify with BAF57 off an anti-BAF57 immunoaffinity column. The start material (Start) was DNA-cellulose-purified PYR complex. The vast bulk of the protein loaded onto the column does not bind (FT). This was confirmed by silver staining (data not shown). The column was washed twice with 0.1 M NaCl buffer (0.1 A and B) and twice with 0.5 M NaCl buffer (0.5 A and B) and then eluted with 0.1 M glycine (pH 2.5) (Elute). BAF57 and Ikaros, particularly Ikaros isoform 2, bind very tightly to the column, withstanding the 0.5 M salt washes. Both BRG1 and Mi-2 bind to the column with somewhat lower affinity, eluting in the 0.5 M salt and pH 2.5 glycine fractions. (C) Ikaros, NuRD, and SWI/SNF proteins copurify with Mi-2 off an anti-Mi-2 immunoaffinity column. The start material (Start) was DNA-cellulose-purified PYR complex. Silver-stained gels showed that the vast bulk of the protein loaded onto the column does not bind (data not shown). The column was washed and eluted as described for panel B. Mi-2 binds very tightly to the column, with much of the Mi-2 remaining on the column after the pH 2.5 elution step. HDAC2, Ikaros isoforms 1 and 2, and SWI/SNF proteins (BAF57, BRG1, and BAF155) bind to the column with high affinity, eluting only in the pH 2.5 glycine fraction.