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. 2008 Nov 24;29(3):714–725. doi: 10.1128/MCB.00825-08

FIG. 1.

FIG. 1.

CTCF is SUMOylated by SUMO 1, 2, and 3, and SUMOylation depends on the SUMO protein's terminal diglycine residues. (A) CTCF is SUMOylated by SUMO 1, 2, and 3. HEK 293 cells were transfected with plasmids encoding FLAG-tagged CTCF and one of the three SUMO proteins or the ubiquitin-like protein Ufm1, all tagged with the myc epitope, as indicated above the top blot. (Top) The whole-cell lysates were immunoprecipitated (IP) with an antibody directed against the FLAG epitope (for CTCF), analyzed by SDS-PAGE and Western blotting, and probed with anti-myc antibody to detect SUMO or Ufm1 modifications. CTCF (>170,000-molecular-weight band) is modified by SUMO 1, 2, and 3 (lanes 2, 3, and 4) but not by Ufm1 (lane 5). Single transfections with CTCF and SUMO 1, 2, or 3 plasmids gave no signal. (Bottom) The same blot was stripped and reprobed with anti-FLAG antibody to show that CTCF was efficiently immunoprecipitated (lanes 1 to 5), whereas no signal was detected after transfection by SUMO- or Ufm1-encoding plasmids alone (lanes 6 to 9). (B) Endogenous CTCF is SUMOylated by SUMO 1. HEK 293 cells were harvested, lysed, and immunoprecipitated with antibody against SUMO 1 or mouse IgG as a control. The immunoprecipitates were analyzed by SDS-PAGE and Western blotting. Probing with anti-CTCF antibody detected a high-molecular-weight form of CTCF modified by SUMO 1 (lane 2). Whole-cell lysate (WCL) (0.85% of the input into each immunoprecipitation) was run in lane 1 to show the unmodified form of CTCF, which migrates faster through the SDS-PAGE gel than the SUMOylated form of CTCF (lane 2). (C) SUMOylation of CTCF by SUMO 1 and 2 is dependent on the two C-terminal glycine residues. HEK 293 cells were transfected with the plasmids indicated at the top. (Top) Whole-cell lysates were immunoprecipitated with anti-FLAG antibody, and Western blots were probed with anti-myc antibody. SUMOylated CTCF was detected after transfection with wild-type SUMO plasmids (lanes 2 and 4) but was absent or greatly reduced with the GG→AA mutant constructs (lanes 3 and 5). The bands were absent after transfection with single plasmids (lanes 1, 6, and 7). (Bottom) Probing the same blot with anti-FLAG antibody (for CTCF) showed that CTCF was efficiently immunoprecipitated.