Identification of NFY as a component of protein complex “R.”

Nuclear extracts from HeLa, BAE, and BSMC cells (5 μg) were incubated with IgG antibody, anti-E2A, or anti-NF-YA antibodies (1 μg each) for 3 hours at 4°C prior to addition of the labeled probe. The probe used was the same as described in Figure 2. After addition of the labeled probe (10 000 cpm/reaction), reaction mixtures were incubated at room temperature for an additional 30 minutes and analyzed on a nondenaturing acrylamide gel as described in Figure 2. Lane 1 represents the probe alone with no nuclear extracts. Lanes 2 to 13 represent probe incubated with nuclear extracts in the presence (+) or absence (−) of each antibody. The positions of the NFY and supershifted complexes (shown as SS) are shown by the arrows. The figure shown is a representative of 2 independent experiments.