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. 2016 Mar 4;7:10830. doi: 10.1038/ncomms10830

Figure 3. PTEN–SRF interact with CArG boxes of SM genes.

Figure 3

(a) Chromatin immunoprecipitation (ChIP) analyses for protein binding to the Myh11 or Acta2 promoters. DNA from serum-restricted rat (left) or human (right) SMCs was cross-linked with formaldehyde and recovered from immunoprecipitated samples using SRF, PTEN or Elk-1 (rat SMCs only) antibodies or a no-antibody negative control. Immunoprecipitated DNA was subjected to qPCR amplification using primers flanking essential CArG boxes in the Myh11 and Acta2 promoters. About 2% genomic DNA input was used as a positive control. (b–e) Electrophoretic mobility shift assays (EMSAs) were conducted as described in Methods using the indicated amount of a fluorescently labelled 20-bp DNA fragment containing CArG ‘B' of the Acta2 promoter (b) or a 95-bp DNA fragment consisting of CArGs ‘A' and ‘B' of the Acta2 promoter (c–e) and the indicated volumes of purified recombinant SRF and PTEN. (b) EMSA with 20-bp DNA fragment. Positions of SRF-containing complexes are labelled 1 and 2 (lane 2); positions of SRF–PTEN-containing complexes are labelled 3 and 4 (lane 3); unbound DNA not shown. (c) EMSA with 95-bp DNA fragment. DNA plus rSRF alone (lane 2) compared with DNA plus rSRF and increasing amounts of rPTEN (lanes 3–6). Lane 7 shows DNA plus rPTEN alone. (d) EMSA with 95-bp DNA fragment. DNA plus 1 μl rSRF (lane 2) or saturating amounts (3 μl) of rSRF (lane 5) compared with DNA plus 3 μl rPTEN and 1 μl rSRF (lane 3) or saturating amounts (3 μl) of rSRF (lane 4). Position of SRF-containing complexes are labelled ‘2' (lane 5); position of SRF–PTEN-containing complexes are labelled ‘1' (lane 4). (e) EMSA with 95-bp DNA fragment. DNA plus 1 μl rSRF (lane 2), DNA plus 3 μl rPTEN and 1 μl rSRF (lane 3), DNA plus 3 μl rPTEN and 1 μl rSRF (lane 3) supershifted with a PTEN-specific antibody (lane 4), and DNA plus 3 μl rPTEN and 1 μl rSRF (lane 3) supershifted with an SRF-specific antibody (lane 5). Position of SRF–PTEN-containing complexes are labelled ‘1', position of SRF–PTEN-containing complexes supershifted with a PTEN antibody are labelled ‘3' and position of SRF–PTEN-containing complexes supershifted with an SRF antibody are labelled ‘2'. Shown for each panel are representative images from a minimum of three independent experiments. qPCR, quantitative PCR.