Figure 3: Arid5b directly regulates Col2a1 expression.

(a) Schematic presentation of the putative Arid5b-binding element (CATAT) and primers used for ChIP assays in the promoter region of mouse Col2a1. (b,c) ChIP assays. C3H10T1/2 cells were infected with Venus or Venus-Arid5b adenovirus and then immunoprecipitated with anti-GFP antibodies. The binding of Arid5b to the Col2a1 promoter region was examined by PCR using the primers shown in (a). The binding of Arid5b to the Col2a1 promoter region was also examined by real-time PCR. Data are shown as percentages of input (mean±s.d., n=3) *P<0.01 (versus Venus); Student’s t-test. (d) DNA pull-down assays using the Arid5b-binding element in the Col2a1 promoter. Lysates of 293FT cells transfected with empty or Flag-Arid5b vector were incubated with biotinylated oligonucleotide containing a putative Arid5b-binding element in the Col2a1 promoter in the presence or absence of the unlabelled oligonucleotide (competitor). After precipitation with streptavidin beads, proteins associated with the oligonucleotide were determined by immunoblotting with an anti-Arid5b antibody. (e) Mutation analysis of the Arid5b-binding element in the Col2a1 promoter. C3H10T1/2 cells were transfected with a WT or mutant Col2a1–luciferase construct together with the indicated expression vectors. Luciferase activities were measured 48 h after transfection. Data are expressed in relative luciferase units (mean±s.d., n=3). *P<0.01; two-way ANOVA followed by a Tukey’s HSD post hoc test.