FIGURE 2.

Motif 1 EMSAs. A, motif 1 (M1C and M1T) probes incubated with pA80BR NE. The EMSA was performed twice per sheep cell extract (pA80BR, sA80BR, IS120Cer, IS120Liv); only data for pA80BR are shown. The intensity of the M1C-protein complex was consistently as high as shown, whereas the M1T-protein complex varied between 0 and 10% that of the M1C complex (indicated with >). B, competition EMSA; Lanes 1–6, variant motif 1 probe (M1C) incubated with pA80BR NE in the presence of anti-YY1 antibody (Ab) and unlabeled probes as indicated; lanes 7–11, control experiment: motif 2 probe (M2C) incubated with pA80BR NE in the presence of anti-YY1 antibody (Motif 2 specific complex indicated with ≫). The antibody competition assay was performed a total of 4 times in different combinations as shown in B–D. C, competition EMSA. Variant motif 1 probe (M1C) incubated with pA80BR NE in the presence of increasing concentrations of anti-YY1 antibody. D, tissue extracts (performed twice with comparable results). Motif 1 probe (M1C) incubated with ovine pA80BR NE as control, ovine and murine brain tissue-derived Nes, and murine spleen tissue-derived NE. Specific complexes I (Fy-I), IIA and IIB (Fy-II), and III (YY1) were detected in tissues. The anti-YY1 antibody was added immediately after the addition of ³²P probe and incubated at room temperature for 45 min. Competitor, unlabeled probe M1C; Non-competitor, unlabeled probe NC-1. 10×, 100×, 200×, or 1000× indicates the addition of a 10, 100, 200, or 1000-fold molar excess of unlabeled probe. *, unbound ³²P-labeled probe.