Figure 6.
SUMO modification of shuttling Med. (A) 293 cells were transfected with the indicated plasmids and His6-SUMO conjugates were purified by nickel affinity chromatography. Western blots show the levels of SUMOylated HA-tagged Med (top panel) and associated pMad protein (bottom panel). Arrow indicates SUMO-modified Med. (B) SUMO assay as described in A with the indicated transfected plasmids. MedG727D is compromised with respect to its ability to interact with Mad and enter the nucleus, whereas the Mad3SA mutant cannot be phosphorylated or enter the nucleus following pathway activation. (C) Detection of SUMO-modified Med as described in A. Lanes 1–3 are all taken from the same exposure of the same Western blot; the gap reflects only the removal of irrelevant lanes.