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. 2015 Apr;27(2):148–155. doi: 10.3978/j.issn.1000-9604.2015.04.03

Figure 2.

Figure 2

Kaiso can bind to methylated- and hydroxymethylated-CGCG sequence in vitro. (A) Purification of IPTG-induced GST-Kaiso from E. coli. GST-Kaiso and GST control were both detected using Commassie Brilliant blue staining (left image) and Western blotting with the anti-GST antibody (right image); (B) Results of EMSA assay show that GST-Kaiso can bind to the biotin-labeled synthetic 5-methylcytosine probe (mC probe; lane #6) and 5-hydroxymethylcytosine probe (hmC probe; lane #13), but not to the unmethylated cytosine probe (C probe; lane #3). In the competitive EMSA assays, interaction between Kaiso and mC or hmC probe is completely impaired by Kaiso antibody (lane #7 or #16) or the unlabeled mC probe pretreatment (lane #9 or #15), but not by the unlabeled and unmethylated C probe pretreatment (lane #8). The unlabeled hmC probe inhibits Kaiso-hmC interaction completely. However it only partially impairs Kaiso-mC interaction. EMSA, electrophoretic mobility shift assay.