Figure 6
Binding of PAX1 to the 3′ region of the UE. (a) A 359-bp DNA sequence of the mouse UE. The SOX9-binding sites are underlined. (b) ChIP assays on extracts from AF cells. After immunoprecipitation of the cross-linked extracts with anti-PAX1 antibody, anti-SOX9 antibody, or normal IgG antibody, the DNA was subjected to PCR with primers that amplify a 116-bp fragment of the UE, 216-bp fragment of the I12E, 135-bp fragment of Nkx3.2-P, or 328-bp fragment of the Col2a1-E. Full-length gels are presented in Supplementary Fig. 7. (c,d) Gel shift assays were performed using 20 fmol of biotin-labelled dsDNA probes, 20 pmol of unlabeled dsDNA fragments, and nuclear extracts (N.E.) of HEK293T cells transfected with pcDNA3 empty vector (N.E. Control) or pcDNA3-FLAG-Pax1 (N.E. FLAG-PAX1). The biotin-labelled and unlabeled DNA fragments correspond to positions 1–130, 118–259, and 249–359 of the UE. A shifted band was caused by interactions between FLAG-PAX1 and the biotin-labelled 249–359 of UE. The shifted band in (c) disappeared in the presence of the unlabeled 249–359 oligonucleotide or anti-FLAG antibody (Antibody). The shifted band in (d) disappeared in the presence of the unlabeled 315–359 oligonucleotide but remained detectable in the presence of the unlabeled 249–290 or 281–325 oligonucleotide. (e,f) Gel shift assays were performed using biotin-labelled oligonucleotides shown in Supplementary Table 1 and nuclear extracts (N.E.) of HEK293T cells transfected with pCAG empty vector (N.E Control), pCAG-Sox9 (N.E. SOX9), or pCAG-Pax1 (N.E. PAX1). For the assays in (e,f), 100 and 5 fmol of the biotin-labelled oligonucleotides were used, respectively. A total of 1.5 μL of N.E. was used for each binding reaction in (f). A shifted band is shown with an arrow. Non-specific bindings are shown with open arrowheads. s, shifted band.
