Fig. 2

Physical and functional interaction between Dlx and Smad transcription factors. A, In vivo co-immunoprecipitation. Extracts obtained from dissected E15.5 telencephalon were subjected to immunoprecipitation with anti-phosphorylated Smad2 (lane 4) or control (lane 2 and 3) antibodies, as indicated. Immunoprecipitates, together with 1% of input lysate (lane 1), were subjected to Western blotting with a chicken anti-Dlx2 antibody. The position of migration of the immunoglobulin light chain (IgG LC) is indicated. B, Chromatin immunoprecipitation. Protein-DNA complexes from dissected E15.5 telencephalon were subjected to immunoprecipitation with the indicated antibodies, followed by PCR with oligonucleotide primers specific for the Dlx1/2, Dlx5/6 and Arx enhancers or the p21^Cip1 distal promoter. Dlx2, Smad1, Smad2/3, and Smad4 were associated with the Dlx1/2, Dlx5/6 and Arx enhancers, but not with the distal region of the p21^Cip1 promoter. Total genomic DNA was used as a positive control (Input). C, Transient transcription assays. The effect of Smad1, Smad2 or Smad4 on Dlx1-, Dlx2- and Dlx5-mediated transcriptional activity was assessed by co-transfecting COS cells with a LacZ reporter plasmid driven by the Dlx5/6 enhancer-i (mI56i-LacZ). Results are shown relative to the activity of the reporter alone and represent the means ± the S.D. of at least four experiments performed in duplicates (*, p < 0.05 using one-way analysis of variance with a Dunnett’s post test). A reporter plasmid containing the luciferase gene under the control of the RSV promoter was used as a control for transfection efficiency