Figure 5.
Med is SUMO modified in the nucleus. (A) The relative proportion of Med protein in the cytoplasmic (C) and nuclear (N) fractions of 293 cells transfected with the indicated plasmids is detected by Western blotting with an anti-HA antibody. (B) The indicated plasmids were transfected into 293 cells, followed by nickel affinity chromatography of His6-SUMO conjugates. (Top panel) SUMO modification of HA-tagged wild-type Med protein was determined by Western blot analysis of the nickel eluates. The arrow points to SUMO-modified Med. (Bottom panel) Western blot showing the increase in Mad following its titration. (C) 293 cells were transfected with Mad, Med, His6-SUMO, and Tkv-QD plasmids, and where labeled (+) treated with Leptomycin B (LMB), an inhibitor of Smad4/Med export. SUMO conjugates were purified, and the amount of SUMO-modified Med in whole-cell extracts (left panel) or nuclear and cytoplasmic fractions (right panel) was visualized by Western blot analysis. Arrow indicates SUMOylated Med.