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. 2021 Jun 19;297(1):100901. doi: 10.1016/j.jbc.2021.100901

Figure 5.

Figure 5

EMSA analysis demonstrating that binding of Alx1 to closely spaced half sites is noncooperative.A, all probes used were 70 bp in length and included the Sp-EMI/TM palindromic site or two half sites (site A and site B) from the Sp-mtmmpb CRM. Additional nucleotides underlined were inserted between site A and site B to increase the distance between the two sites and alter their relative orientation on the DNA helix. The added sequence was designed by repeating the sequence underlined in the probe containing the two half sites to synthesize site A +++ site B probe. Binding specificity was confirmed by adding WT or mutant competitor. B, protein titration of Alx1-FL with constant amounts of two different Cy5-labeled probes containing sites A and B. C, quantification of the representative gel in panel B. Plot of the fraction bound as a function of protein concentration of two independent replicates with the data represented as the mean ± SD. The filled squares denote the probe site A + site B, and the empty squares denote the probe site A +++ site B. For the complete sequence of all probes used in this study, see Table S1. Alx1-FL, full-length Alx1; CRM, cis-regulatory module; F, free probe.