FIG. 6.

The SUMO E3 ligase Pc2 increases the SUMOylation of CTCF. HeLa cells were transfected using Fugene 6 transfection reagent with the indicated plasmids. The whole-cell lysates were precleared with anti-T7-agarose resin prior to immunoprecipitation (IP) with anti-FLAG antibodies. After SDS-PAGE and Western blotting, the membrane was probed with anti-myc antibody to detect SUMO 1, 2, or 3 (top) or with anti-FLAG antibody to detect CTCF (middle). SUMO E3 ligase activity was indicated by the increase in the SUMOylation signal in lanes 3 and 11. Pc2 exhibited no SUMO E3 ligase activity for the SUMOylation of CTCF by SUMO 1 (lane 6). CTCF degradation products were also SUMOylated by SUMO 2 and 3 in the presence of Pc2. Flowthrough from the immunoprecipitation was blotted for β-actin to show equal loading (bottom).