<p>(<b>A</b>) Relative amount of <i>HSFA2</i> RNA determined by quantitative RT-PCR in three week old WT (Col-0), WT2 (Ws), triple and quadruple <i>HSFA1</i> mutant seedlings. Bars represent the <i>HSFA2</i> transcript in relation to <i>AtSAND</i> and normalized to Col-0 WT at 0 h HS. Error bars correspond to the s.e.m. (n = 3). (<b>B</b>) Electrophoretic mobility shift assay (EMSA) with increasing amounts (lanes 1–5) of recombinant HSFA2 protein and constant amounts of radioactively labeled <i>ONSEN</i> LTR fragment derived from <i>ONSEN 1</i>. Competition by unlabeled DNA of the full length LTR in 50-fold excess (lane 6) or the fragment containing only the heat-responsive element (Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004115#pgen.1004115.s007" target="_blank">Table S2</a>) in a 200-fold excess (lane 7) demonstrate the binding specificity. (<b>C</b>) Relative amount of <i>ONSEN</i> RNA determined by quantitative RT-PCR in wild type and <i>hsfa2-1</i> mutant plants after 30 h HS. Bars represent the <i>ONSEN</i> transcripts in relation to that of <i>AtSAND</i> and normalized to WT at 0 h HS. Error bars correspond to the s.e.m. (n = 3). (<b>D</b>) Quantification of <i>ONSEN</i> extrachromosomal DNA from the same samples as in C. Bars represent the ratio between extrachromosomal and integrated copies determined by densitometry after Southern blot analysis. Error bars correspond to the s.d. (n = 3).</p
