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. 2015 Jan 7;290(6):3269–3276. doi: 10.1074/jbc.C114.601906

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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — PICH is enriched at the centromere independently of mitotic SUMOylation and is SUMOylated on mitotic chromosomes. a, PICH is enriched at the centromere, co-localizing with CENPA and SUMO2/3, and the inhibition of SUMOylation by dnUbc9 does not alter PICH localization at the mitotic chromosomes. Replicated mitotic chromosomes were isolated from XEE and subjected to immunofluorescence staining with the indicated antibodies. Cont., control. b, PICH^hs was enriched at the centromere, similar to endogenous PICH on mitotic chromosomes isolated from XEE. PICH^hs-EGFP mRNA was added to XEE with and without the addition of dnUbc9 and was visualized on mitotic chromosomes along with CENPA and SUMO2/3. c, PICH is posttranslationally modified on mitotic chromosomes in XEE. Non-replicated mitotic chromosomes were isolated from XEE and analyzed by immunoblotting with an anti-PICH antibody. SUMOylation was inhibited by the addition of dnUbc9. d, PICH is SUMOylated on mitotic chromosomes in XEE. SUMO2GG-EGFP was added to the XEE, and non-replicated mitotic chromosomes were isolated and analyzed by immunoblotting. PARP1, a known SUMO substrate, served as a positive control for the PARP1-SUMO-EGFP supershift (indicated with asterisks).