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. 2009 Dec 24;285(8):5266–5273. doi: 10.1074/jbc.M109.088088

FIGURE 2.

FIGURE 2.

K-bZIP is a SIM-containing SUMO E3 ligase. A, 293T cells were co-transfected with the indicated plasmids. Forty-eight hours after transfection, TCLs were prepared for co-immunoprecipitation assay with anti-FLAG M2 beads followed by immunoblotting with anti-K-bZIP antibody. B, purified F-K-bZIP was incubated in a sumoylation reaction containing purified E1-activating and E2-conjugating enzyme, in the presence of either SUMO-1, -2, or -3. Immunoblotting with anti-K-bZIP antibody was then used to reveal the presence of SUMO-conjugated K-bZIP. C, in vitro sumoylation of p53 and Rb but not KAP-1 and histone H2B by K-bZIP. Purified p53, Rb, KAP-1, and histone H2B were incubated in a sumoylation mix containing purified E1 activation and E2-conjugating enzyme together with either SUMO-1, -2, or -3 in absence or presence of recombinant wild-type K-bZIP or its L75A mutant. Immunoblotting with anti-p53, anti-Rb, anti-KAP-1, or anti-histone 2B antibody was then used to reveal the SUMO-conjugated proteins. Equal amounts of wild-type F-K-bZIP and its L75A mutant were confirmed by gel electrophoresis and Coomassie Blue staining (upper panel).