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. 2010 Jan 11;285(11):8408–8421. doi: 10.1074/jbc.M109.064386

FIGURE 7.

FIGURE 7.

DNA binding, protein interaction, and luciferase assays using Oct25 mutants. A, EMSAs (8% PAGE) show that the GST-Oct25 fusion protein binds to a canonical octamer motif (underlined) within the Xnr1 promoter. Binding is also observed for the mutant containing only the POU and Hox domains (Oct25PH). Deletion of the Hox-specific domain results in loss of DNA binding activity. GST alone was used as control. The mutants with a truncation of either the complete POU-specific domain (aa 237–301) or different regions of the POU-specific domain lose their DNA interaction capacity. DNA binding is also lost, when the amino acids TTIC are changed to ICTT (TTIC → ICTT) or Cys-274 is mutated to Pro (C274P) but is retained in the (C274S) mutant. B, GST pulldown assays show that Oct25(C274P) and Oct25(ΔPOU(273–301) still interact with Smad4, Smad2, Smad3, Smad1, FAST1, VegT, or TCF3. C, luciferase reporter activities driven by the wild type Xnr1 promoter (Xnr1Luc(−279) or the indicated deletions (15) in the absence or presence of Oct25ΔPOU(273–301). D, luciferase reporter assays driven by the −279(−TCF-Tbox) Xnr1 promoter upon co-injection with Oct25ΔPOU(−273–301) and dnXAR1. E, EMSA of the −279/−5 Xnr1 promoter fragment (6% PAGE) reveals binding of Oct25 but no interaction with Oct25ΔPOU(−273–301) GST fusion proteins.