Fig. 5.

SUMOylation enhances Sf1 binding to Dax1, but does not influence its binding affinity to DNA. (A) SUMOylation increases the binding of Sf1 to Dax1. HEK293T cells were plated at 10⁶ cells/dish in 10 mm cell culture dishes 24 h before transfection and were transfected with 1 µg pcDNA-Sf1 and 1 µg pcDNA-Dax1 vectors. After 48 h, cells were lysed and co-immunoprecipitation assays performed. Lysates were precipitated using anti-Sf1 antibody and immunoblotted with anti-HA (for Dax1) or -Sf1 antibodies. The data were normalized to input and shown as fold change over wild-type Sf1. *P<0.05. Error bars indicate s.e.m. (B) SUMOylation or Dax1 do not change the DNA-binding capacity of Sf1. HEK293T cells were plated at 10⁶ cells/dish in 10 mm cell culture dishes 24 h before transfection and were transfected with 1 µg pcDNA-Sf1 and 1 µg pcDNA-Dax1 vectors with 25 ng of linearized pGL3-FAdE construct. After 48 h, cells were fixed and processed for ChIP assay using anti-Sf1 antibody. The data were normalized to values obtained for 1% input controls, and the results are presented as percentage of input (n=5). Error bars indicate s.e.m.