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. 2007 May 3;35(10):3453–3464. doi: 10.1093/nar/gkm239

Figure 3.

Figure 3.

hStaf/ZNF143-binding assays on wild-type and mutant versions of the BUB1B promoter. (A) Gel retardation assay with a fragment encompassing positions −342/−195 of the BUB1B promoter. The 32P labeled DNA (probe I) was incubated in the absence (lane 1) or presence of increasing amounts of hStaf/ZNF143 DBD (lanes 2–5). The reactions in lanes 6 and 7 were performed with the same amount of protein as in lane 5 but in the presence of a 1000-fold excess of unlabeled specific (wt SBS) and unspecific competitors (unsp). Binding assays in lanes 1–5 and 6, 7 were performed in separate experiments. C1 and C2: complexes containing one and two proteins, respectively. (B) A 148-bp 5′ end-labeled fragment containing the wild-type or mutant versions of the SBS was used in the binding studies. Lanes 1–4, no protein added. Lanes 5–8 binding assays with the same amount of DBD. Probes are indicated above the lanes. (C) The wt (lanes 1–5) or the SBS1 and SBS2 mutant version borne by probe I (lanes 6 and 7) were incubated in the absence (lanes 1 and 6) or presence of increasing amounts of rabbit reticulocyte lysate containing full-length hStaf/ZNF143 (lanes 2 and 3). Lane 7 contained the same amount of protein as in lane 3. Lanes 4 and 5 were performed with the same amount of protein as in lane 3 but in the presence of a 1000-fold excess of unlabeled specific (lane 4, wt SBS) and unspecific competitor (lane 5, unsp), respectively. C1 corresponds to both sites of the probe saturated with the protein; the protein binds to one of the sites only in C2. (D) Gel retardation assay with the wt probes II (lanes 1–8) or III (lanes 9–14) in the absence (lanes 1 and 9) or presence (lanes 2–8 and 10–14) of HeLa cell nuclear extracts (NE). The reactions in lanes 3, 4 and 11, 5, 6 and 12, 7 and 13, 8 and 14 were performed in the presence of unlabeled specific competitor (wt SBS), unspecific competitor (unsp), anti-hStaf/ZNF143 and pre-immune antibody, respectively. The specific competitor was added at a 500-fold (lane 3) and 1000-fold molar excess (lanes 4, 11). A 500 and 1000-fold molar excess of unspecific competitor was contained in lanes 5 and 6, 12 respectively. The arrow points to the complexes mentioned in the text.