Figure 3.

Cloning of Fbxl10, which encodes a protein that binds the highly structured Hoxb9 promoter fragment. (A) Scheme of cloning strategy and schematic diagram of the obtained Fbxl10 gene. Cloning was done by using South–western methods and fragment A as a probe. The 55 clones we isolated were counter-selected by South–western methods and fragment B as a probe. Further confirmation was carried out with EMSA. We obtained the partial sequence of Fbxl10 through South–western cloning. Sequence analysis of full-length Fbxl10 cDNA revealed several motifs: jmjC, cxxc, Zn-finger, PHD Zn-finger and F-box domains and leucine-rich repeats (LRR). (B) EMSA with GST-fusion protein derived from the cloned fragment. The fragment used to produce the GST-fusion protein is indicated as a bold black line located below Fbxl10 (panel A). Competition assays were carried out as described in the legend of Figure 2. As observed with our EMSA analyses performed with nuclear extracts (NE) (see Figure 2), fragment A competed successfully for binding, while fragment B did not. Arrow points to the slow-moving band; asterisk marks the band representing linear DNA.