FIG. 3.
PU.1 and pRB colocalize with GATA-1 at GATA-1 binding sites in chromatin. (A) U2OS cells were transfected as described in the legend to Fig. 1A, and ChIP was carried out as described in Materials and Methods with anti-pRB antibody or an anti-rabbit immunoglobulin G antibody that has the same isotype (Control Ab). PCR assays were performed as described in Materials and Methods with specific primers that differ from the primers used in Fig. 1A; here the presence of the 110-bp fragment indicates the presence of the αD3 promoter region in the immunoprecipitate. Lane 6 shows PCR amplification of αD3-Luc plasmid DNA with the primers. (B) ChIP was performed on cross-linked chromatin from MEL cells as described in Materials and Methods with antibodies to GATA-1, PU.1, and pRB (RB) and with β-tubulin (Mock). The amounts of specific promoter DNA fragments relative to G6PD DNA in duplicate immunoprecipitates were quantitated by real-time PCRs, and the degree of enrichment of the promoter fragments was calculated. Error bars indicate the standard deviation. Panels on the right show the dissociation curves of the set of amplified DNA fragments for HS2 (Tm = 85°C), G6PD (Tm = 78.5°C), and Ey (Tm = 82°C).