FIG. 3.

SUMOylation of CTCF does not affect its DNA binding ability. (A) In-vitro translated ³⁵S-labeled CTCF was incubated in a SUMOylation reaction without (−) and with (+) SUMO 1 protein and run on an SDS-PAGE gel. In the presence of SUMO 1, all of the CTCF was SUMOylated and shifted into more slowly migrating species (the arrows show the modified and unmodified forms of CTCF). Similar experiments also showed efficient SUMOylation by SUMO 2 and 3 (not shown). (B) CTCF was made by in vitro transcription/translation (IVT) and used in electrophoretic mobility shift assays with or without subsequent incubation in an in vitro SUMOylation reaction. The PCR fragments used in each experiment are shown and are described in Materials and Methods and Table 2. Lanes A, IVT mixture without CTCF mRNA; lanes B, IVT using CTCF mRNA; lanes C, IVT CTCF incubated in SUMOylation reaction mixture but without SUMO protein; lanes D, E, and F, IVT CTCF incubated in SUMOylation reaction mixture with SUMO 1, 2, or 3, respectively. U, unbound DNA; B, bound DNA. Careful inspection of lanes containing the SUMO proteins revealed a slight slowing of the shifted band, presumably due to SUMOylation of CTCF. Competition experiments showed that SUMOylation might affect more weakly binding sites (data not shown).