2Δ. The first set of probes tested by EMSA included Bs2, Bs8.1, Bs8.1Δ, Bs8.2Δ and Bs10 (Figure MLN2238 molecular weight 1, Table 1). Bs2 and Bs10 resulted negative (data not shown), while the overlapping oligonucleotides Bs8.1, Bs8.1Δ and Bs8.2Δ (nt -134 to -103) formed GANT61 supplier intense shifted bands that were specifically inhibited with 100-fold excess of cold homologous probes, suggesting specificity (Figure 2A). Oligonucleotides Bs8.1Δ and Bs8.2Δ (nt

-134 to -113) included substitutions at positions -120 (T/A) and/or -104 (C/G) that are characteristic of P. brasiliensis isolates belonging to phylogenetic species PS2, which is presently represented by Pb3 [3, 15]. However, these substitutions did not seem to alter the intensity of protein binding (Figure 2A). In addition, probes Bs8.1, Bs8.1Δ and Bs8.2Δ cross-competed (Figure 2B). The Bs8.1, Bs8.1Δ and Bs8.2Δ complexes migrated similarly and the probes are similar in size signaling pathway (22 and 24 mer), suggesting binding to the same protein. Therefore, our results point to a protein binding core in the overlapping sequence TGCAGAA/TTTATCAA. Alternatively,

all the probes are competing for distinct Sox-5-like protein binding sites (Figure 1). It is necessary to point out, however, that all the interpretations drawn from EMSA using total protein extracts will only possibly be confirmed by using either purified transcription factors or specific antibodies in super-shift experiments, considering that differences in shifts could be evoked by the same protein, while similar migrations could alternatively be the result of different transcription factors. Figure 2 Radioautograms showing EMSA results with Pb339 protein extracts and radio labeled (*) Bs8.1, Bs8.1Δ, and Bs8.2Δ probes. In A, specificity of the EMSA

bands was suggested by effective competition with 100 × molar excess of cold homologous probe. In B, cross-competition experiments with the indicated probes. Molar Telomerase excess of cold competitors was 100 ×. The position of shifted bands is indicated with arrows. The next set of probes tested by EMSA included Et12, Et23, Et23Δ, Et4 and Et5 (Figure 1, Table 1). We tested these regions based on apparent protection in DNAse I protection footprinting assays (data not shown). In EMSA, probes Et4 and Et5 formed only weak and unspecific complexes with P. brasiliensis total protein extracts (data not shown), although these regions are rich in predicted transcription elements (Figure 1). We also tested an Et4 variant that had five extra upstream nucleotides. EMSA results were still negative, suggesting that the NIT2 motif predicted in this probe (Figure 1) is not functional. Overlapping Et12 and Et23 oligonucleotides (nt -255 to -215) formed intense complexes that co-migrated and could be specifically inhibited with 100-fold excess of cold homologous probe (Figure 3A).