In addition, an auxiliary check was performed against structured Pfam domains

h figure, and taking the mean and SD of three independent experiments for each assay. Statistical significance of the difference was calculated by t test of the means. The XEE pull-down assays were performed as described previously with 10 mM iodoacetamide addition in buffers to UNC0642 web prevent 675 DNA topoisomerase II regulates H3 kinase Haspin Yoshida et al. deSUMOylation activity in the XEEs. XEEs were diluted by two times PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19837474 their volume with PD buffer, and diluted XEEs were centrifuged at 25,000 g for 45 min at 4C. An equal volume of the PD buffer supplemented with 0.2% Tween 20 and 0.2% Triton X-100 was added to the supernatants and incubated with S-tagged TOP2A CTD-bound or SUMOylated TOP2A CTD-bound S-agarose beads for 1 h at RT. After washing with PD buffer, the beads were incubated in the dilution buffer containing 35 g/ml SENP2-CD for 45 min at RT to cleave conjugated SUMO2 from TOP2A CTD and dissociate pulled-down proteins from the beads. SDS-PAGE samples were prepared by adding a half volume of 3 SDS-PAGE sample buffer to the bead suspension. All samples were separated on 816% Trisglycine gels by SDS-PAGE and analyzed with silver staining or immunoblotting. For the preparation of samples for LC/MS-MS analysis, pull-down samples and the soluble fractions were isolated using spin columns, washed with urea, and precipitated with trichloroacetic acid. Samples were subjected to LC/MS-MS analysis for protein identification. In the case of pull-down assays with HaspinGFPexpressing XEEs, interphase XEEs with 10 ng/l Haspin-GFP mRNA were incubated for 150 min at RT and then returned to mitotic phase by adding an equal volume of CSF XEEs or kept in interphase XEEs for the pull-down experiment in Fig. 6 A. Protein expression levels in XEEs after incubation with mRNA were checked through immunoblotting and adjusted accordingly with additional volumes of the original CSF XEEs or interphase XEEs to achieve similar protein expression concentrations before being used for pull-down assays. Immunoblotting signals were acquired by Image Station 4000R, and signal levels were quantified by ImageJ software. Relative levels were calculated by measuring the signal levels of each protein band, normalizing the values to the recombinant TOP2A CTD levels, and taking the mean and SD of three independent experiments for each assay. Statistical significance of the difference was calculated by t test of the means. Immunofluorescence analysis of chromosomes GFP mRNA was incubated in interphase XEEs at RT for 60 min at a concentration of 20 ng/l or at multiple concentrations of 20, 40, and 60 ng/l. Afterward, demembraned sperm nuclei were added to allow for DNA replication. After the completion of DNA replication, an equal volume of CSF XEEs was added and incubated for 45 min for mitotic CSF XEEs with Haspin-GFP. DNA was stained with Hoechst 33342 dye, and the samples were mounted using Vectashield H-1000 medium. All images were acquired using the Nikon Plan Apo 100/1.4 oil objective lens on a TE2000-U microscope with a Retiga SRV CCD camera operated by Volocity imaging software at RT. Photoshop CS6 was used to process the obtained images from Volocity to show the signal intensities by adjusting overall intensity range levels equally within independent experiments without any gamma adjustments. Images were cropped and the resolution was adjusted to fit journal policy. Quantification of fluorescent signals was through ImageJ and Photoshop CS6 by measuring the signal in