Figure 4

SOX9 indirect competitive Electromobility shift assays. (A) ClustalW alignment of the −30 enhancer with possible SOX9 binding sites highlighted in red, the Rbpj-κ binding sites are also highlighted by flanking yellow markings, and each site is identified by the name assigned to each region. SOX9 is predicted to interact through SOX9 dimeric and monomeric interactions in the −30 and all Acan enhancer. (B) Indirect competitive shift EMSAs using radiolabelled control of the Col2a1 SOX9 binding site probe incubated with SOX9 protein generated from a SOX9 expression vector, UP is a programmed lane generated by no expression vector to detect any non-specific binding, Ctrl is the unlabelled control competing with the labelled probe to ensure competition is detected, (mt = mutated). Wildtype probes from the −30 sites 2, 3 and 7 competed away the binding of the SOX9 control oligo that suggests these sites bind to SOX9. (C) The +28 has two SOX9 binding sites (1 and 4) although they are weaker than the control oligo. (D) The −62 contains one site (site 5) and the −80 encloses three binding sites, 1-2, 5 and 9 (E). The −80 EMSA consisted of two gels ran simultaneously to allow interrogation of all the possible SOX9 binding sites with mutated oligonucleotides alongside.