Thrombin activates a Y box–binding protein (DNA-binding protein B) in endothelial cells
J. Clin. Invest. Olga I. Stenina, et al. 106:579 doi:10.1172/JCI9075 [Go to this article.]

Figure 4
DNA-binding properties of thrombin-activated dbpB. A truncated dbpB cDNA corresponding in size to the thrombin-activated dbpB from EC extracts was transcribed and translated in vitro, and the reaction mixture was used for EMSA with the ThRE, a Y-box single-stranded, or a Y-box double-stranded consensus oligonucleotide as the probe (a). As a control, the empty pcDNA3 vector was used in a similar reaction. Cytosolic extracts were prepared from untreated and thrombin-stimulated ECs as described. EMSA was performed using the ThRE or consensus Y-box sequence oligonucleotide as a probe (b). The same Y-box consensus probe was used to detect full-length dbpB in nuclear extracts of ECs, and the complex was supershifted with anti-dbpB antibody. Control, in vitro transcription and translation reaction performed with an empty pcDNA3 vector (a) or cytosolic and nuclear extracts from untreated ECs (b); truncated dbpB, truncated (207–amino acid) dbpB transcribed and translated in vitro; thrombin, extract from EC stimulated with thrombin (10 U/m for 2 hours); control + Ab, nuclear extract from untreated ECs preincubated with antiserum against the COOH-terminus of full-length dbpB before the binding reaction with the radiolabeled Y-box consensus probe; ThRE, ThRE probe used in EMSA; Y-box SS, sense single-stranded Y-box consensus probe used in EMSA; Y-box DS, double-stranded Y-box consensus probe used; cytosolic, cytosolic extract; nuclear, nuclear extract.