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    Bio-Synthesis Inc nsl1
    CENP-C is necessary for the assembly of the Mis12/MIND complex and CENP-K. (A) <t>Nsl1</t> localization is independent of CENP-C. Representative immunofluorescence image of Nsl1 and CENP-C localization in mock- or CENP-C-depleted extracts are shown. Bar, 5
    Nsl1, supplied by Bio-Synthesis Inc, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nsl1/product/Bio-Synthesis Inc
    Average 85 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    nsl1 - by Bioz Stars, 2020-05
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    85
    Mimotopes fluorescein labeled nsl1 peptides
    EM analysis of MIS12C–SPC24–SPC25. (A) Negative-stain EM was performed on the recombinant MIS12C <t>NSL1-258</t> –SPC24 57–197 –SPC25 70–224 complex. (B) Class averages from negative-stain EM images shown in A. The arrowheads point to an element of density that is not present in the class averages of the MIS12C NSL1-258 complex shown in Fig. 1 D and that likely represents the SPC24–SPC25 complex. Bars: (A) 10 nm; (B) 5 nm.
    Fluorescein Labeled Nsl1 Peptides, supplied by Mimotopes, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescein labeled nsl1 peptides/product/Mimotopes
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    fluorescein labeled nsl1 peptides - by Bioz Stars, 2020-05
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    Image Search Results


    CENP-C is necessary for the assembly of the Mis12/MIND complex and CENP-K. (A) Nsl1 localization is independent of CENP-C. Representative immunofluorescence image of Nsl1 and CENP-C localization in mock- or CENP-C-depleted extracts are shown. Bar, 5

    Journal: Molecular Biology of the Cell

    Article Title: Dissection of CENP-C-directed Centromere and Kinetochore Assembly

    doi: 10.1091/mbc.E09-05-0378

    Figure Lengend Snippet: CENP-C is necessary for the assembly of the Mis12/MIND complex and CENP-K. (A) Nsl1 localization is independent of CENP-C. Representative immunofluorescence image of Nsl1 and CENP-C localization in mock- or CENP-C-depleted extracts are shown. Bar, 5

    Article Snippet: Peptide antibodies were generated as previously described ( ) against peptides for CENP-K (acetyl-CRHPEDPKRIRLE-amide) and Nsl1 (acetyl-CRPVETTPRETEAKVK-amide) synthesized by Bio-Synthesis (Lewisville, TX).

    Techniques: Immunofluorescence

    EM analysis of MIS12C–SPC24–SPC25. (A) Negative-stain EM was performed on the recombinant MIS12C NSL1-258 –SPC24 57–197 –SPC25 70–224 complex. (B) Class averages from negative-stain EM images shown in A. The arrowheads point to an element of density that is not present in the class averages of the MIS12C NSL1-258 complex shown in Fig. 1 D and that likely represents the SPC24–SPC25 complex. Bars: (A) 10 nm; (B) 5 nm.

    Journal: The Journal of Cell Biology

    Article Title: The MIS12 complex is a protein interaction hub for outer kinetochore assembly

    doi: 10.1083/jcb.201002070

    Figure Lengend Snippet: EM analysis of MIS12C–SPC24–SPC25. (A) Negative-stain EM was performed on the recombinant MIS12C NSL1-258 –SPC24 57–197 –SPC25 70–224 complex. (B) Class averages from negative-stain EM images shown in A. The arrowheads point to an element of density that is not present in the class averages of the MIS12C NSL1-258 complex shown in Fig. 1 D and that likely represents the SPC24–SPC25 complex. Bars: (A) 10 nm; (B) 5 nm.

    Article Snippet: Fixed concentrations (10 nM) of fluorescein-labeled NSL1 peptides (synthesized by Mimotopes) were mixed with increasing concentrations of the indicated SPC24–SPC25 or HP1 constructs in PBS buffer, and reaction mixtures were allowed to equilibrate overnight at 4°C.

    Techniques: Staining, Recombinant

    The C-terminal domain of KNL1 interacts directly with MIS12C. (A) Size-exclusion chromatography analysis demonstrates that MIS12C and KNL1 2106–2316 form a stoichiometric complex. (B) Cross-linking analysis. In lanes 1–3, 5 nmol, 11 nmol, or 22 nmol BS 2 G was added, respectively. (C) Summary of identified intersubunit cross-links. The full list of cross-links is listed in Table S2 . (D) Multiple sequence alignment of the C-terminal regions of NSL1 from different species. The two red dots indicate residues that were mutated into glutamic acid (E) in the NSL1 EE mutant. (E) Size-exclusion chromatography analysis on the interaction of KNL1 2106–2316 with a construct encompassing residues 227–281 of the NSL1 subunit of the MIS12C. Elution profile of KNL1 2106–2316 (top), NSL1 227–281 (middle), and of a stoichiometric combination of KNL1 2106–2316 and NSL1 227–281 (bottom). (A and E) Dashed gray lines and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively. (F) ITC on the KNL1 2106–2316 –NSL1 227–281 interaction reveals a K d of ∼130 nM and a stoichiometry of 1:1.

    Journal: The Journal of Cell Biology

    Article Title: The MIS12 complex is a protein interaction hub for outer kinetochore assembly

    doi: 10.1083/jcb.201002070

    Figure Lengend Snippet: The C-terminal domain of KNL1 interacts directly with MIS12C. (A) Size-exclusion chromatography analysis demonstrates that MIS12C and KNL1 2106–2316 form a stoichiometric complex. (B) Cross-linking analysis. In lanes 1–3, 5 nmol, 11 nmol, or 22 nmol BS 2 G was added, respectively. (C) Summary of identified intersubunit cross-links. The full list of cross-links is listed in Table S2 . (D) Multiple sequence alignment of the C-terminal regions of NSL1 from different species. The two red dots indicate residues that were mutated into glutamic acid (E) in the NSL1 EE mutant. (E) Size-exclusion chromatography analysis on the interaction of KNL1 2106–2316 with a construct encompassing residues 227–281 of the NSL1 subunit of the MIS12C. Elution profile of KNL1 2106–2316 (top), NSL1 227–281 (middle), and of a stoichiometric combination of KNL1 2106–2316 and NSL1 227–281 (bottom). (A and E) Dashed gray lines and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively. (F) ITC on the KNL1 2106–2316 –NSL1 227–281 interaction reveals a K d of ∼130 nM and a stoichiometry of 1:1.

    Article Snippet: Fixed concentrations (10 nM) of fluorescein-labeled NSL1 peptides (synthesized by Mimotopes) were mixed with increasing concentrations of the indicated SPC24–SPC25 or HP1 constructs in PBS buffer, and reaction mixtures were allowed to equilibrate overnight at 4°C.

    Techniques: Size-exclusion Chromatography, Sequencing, Mutagenesis, Construct

    Role of the NSL1 C-terminal tail. (A) The DSN1–NSL1 subcomplex is sufficient to bind KNL1 and NDC80C. GST-NSL1–DSN1 complex was purified by glutathione Sepharose affinity purification and used as an affinity bait for pull-down assays. Copurification of DSN1 with GST-tagged NSL1 was assessed by immunoblotting. GST-NSL1–DSN1 specifically binds KNL1 2106–2316 (lane 4) and NDC80C (as judged by immunoblotting on the NDC80 subunit; lane 5), and the binding is not mutually exclusive (lane 6). The levels of unspecific binding to GST are shown in lanes 1 and 2. (B) HeLa cells were transiently transfected with plasmids containing GFP alone, GFP-tagged NSL1, and GFP-tagged NSL1 EE . After 48 h, cells were washed once and treated with 5 µM S-trityl- l -cysteine for 16 h. Mitotic cells were harvested by vigorous shake-off. Immunoprecipitates (IPs) with an anti-GFP antibody were examined by immunoblotting (IB) with the indicated antibodies. Only GFP-NSL1 was able to bind endogenous NDC80. The band indicated by the asterisk may represent a degradation product of the GFP fusion proteins. wt, wild type. (C) HeLa cells were transiently transfected with plasmids containing GFP alone, GFP-tagged NSL1, GFP-tagged NSL1 EE , and GFP-tagged NSL1 258 . After 48 h, cells were washed once, treated with 3.3 µM nocodazole for 14 h, and analyzed by immunofluorescence. Bar, 5 µm. (D) The means of KNL1 or NDC80 kinetochore (KT) intensities normalized to CREST signal in cells transiently expressing GFP alone, GFP-tagged NSL1, GFP-tagged NSL1 EE , and GFP-tagged NSL1 258 from the experiment in C were calculated and plotted. Error bars represent the SEM. For each condition, at least four different cells were used in the quantification.

    Journal: The Journal of Cell Biology

    Article Title: The MIS12 complex is a protein interaction hub for outer kinetochore assembly

    doi: 10.1083/jcb.201002070

    Figure Lengend Snippet: Role of the NSL1 C-terminal tail. (A) The DSN1–NSL1 subcomplex is sufficient to bind KNL1 and NDC80C. GST-NSL1–DSN1 complex was purified by glutathione Sepharose affinity purification and used as an affinity bait for pull-down assays. Copurification of DSN1 with GST-tagged NSL1 was assessed by immunoblotting. GST-NSL1–DSN1 specifically binds KNL1 2106–2316 (lane 4) and NDC80C (as judged by immunoblotting on the NDC80 subunit; lane 5), and the binding is not mutually exclusive (lane 6). The levels of unspecific binding to GST are shown in lanes 1 and 2. (B) HeLa cells were transiently transfected with plasmids containing GFP alone, GFP-tagged NSL1, and GFP-tagged NSL1 EE . After 48 h, cells were washed once and treated with 5 µM S-trityl- l -cysteine for 16 h. Mitotic cells were harvested by vigorous shake-off. Immunoprecipitates (IPs) with an anti-GFP antibody were examined by immunoblotting (IB) with the indicated antibodies. Only GFP-NSL1 was able to bind endogenous NDC80. The band indicated by the asterisk may represent a degradation product of the GFP fusion proteins. wt, wild type. (C) HeLa cells were transiently transfected with plasmids containing GFP alone, GFP-tagged NSL1, GFP-tagged NSL1 EE , and GFP-tagged NSL1 258 . After 48 h, cells were washed once, treated with 3.3 µM nocodazole for 14 h, and analyzed by immunofluorescence. Bar, 5 µm. (D) The means of KNL1 or NDC80 kinetochore (KT) intensities normalized to CREST signal in cells transiently expressing GFP alone, GFP-tagged NSL1, GFP-tagged NSL1 EE , and GFP-tagged NSL1 258 from the experiment in C were calculated and plotted. Error bars represent the SEM. For each condition, at least four different cells were used in the quantification.

    Article Snippet: Fixed concentrations (10 nM) of fluorescein-labeled NSL1 peptides (synthesized by Mimotopes) were mixed with increasing concentrations of the indicated SPC24–SPC25 or HP1 constructs in PBS buffer, and reaction mixtures were allowed to equilibrate overnight at 4°C.

    Techniques: Purification, Affinity Purification, Copurification, Binding Assay, Transfection, Immunofluorescence, Expressing

    Interaction of MIS12C with NDC80C. (A) Schematic representation of the NDC80C. (B) Binding curves were measured by ITC by titrating NDC80C (left) or SPC24 57–197 –SPC25 70–224 (right) in a cell containing MIS12C NSL1-258 . (C) Size-exclusion chromatography elution profiles and SDS-PAGE analysis of recombinant NDC80C expressed in, and purified from, insect cells (top), MIS12C NSL1-258 (middle), and their stoichiometric combination (bottom). (D) Elution profiles and SDS-PAGE analysis of NDC80C (top), MIS12C NSL1-227 (middle), and their stoichiometric combination (bottom). The MIS12C NSL1-227 construct binds NDC80C. (E) As in C, but with the kinetochore-binding portion of NDC80C, the SPC24 57–197 –SPC25 70–224 construct. (C–E) Dashed gray lines and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively.

    Journal: The Journal of Cell Biology

    Article Title: The MIS12 complex is a protein interaction hub for outer kinetochore assembly

    doi: 10.1083/jcb.201002070

    Figure Lengend Snippet: Interaction of MIS12C with NDC80C. (A) Schematic representation of the NDC80C. (B) Binding curves were measured by ITC by titrating NDC80C (left) or SPC24 57–197 –SPC25 70–224 (right) in a cell containing MIS12C NSL1-258 . (C) Size-exclusion chromatography elution profiles and SDS-PAGE analysis of recombinant NDC80C expressed in, and purified from, insect cells (top), MIS12C NSL1-258 (middle), and their stoichiometric combination (bottom). (D) Elution profiles and SDS-PAGE analysis of NDC80C (top), MIS12C NSL1-227 (middle), and their stoichiometric combination (bottom). The MIS12C NSL1-227 construct binds NDC80C. (E) As in C, but with the kinetochore-binding portion of NDC80C, the SPC24 57–197 –SPC25 70–224 construct. (C–E) Dashed gray lines and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively.

    Article Snippet: Fixed concentrations (10 nM) of fluorescein-labeled NSL1 peptides (synthesized by Mimotopes) were mixed with increasing concentrations of the indicated SPC24–SPC25 or HP1 constructs in PBS buffer, and reaction mixtures were allowed to equilibrate overnight at 4°C.

    Techniques: Binding Assay, Size-exclusion Chromatography, SDS Page, Recombinant, Purification, Construct

    Interaction of MIS12C with HP1. (A) Size-exclusion chromatography elution profiles and SDS-PAGE analysis of MIS12C NSL1-258 (top), HP1-α (middle), and their stoichiometric combination (bottom). (B) The MIS12C NSL1-258EE mutant does not bind HP1-α (top). SPC24 57–197 –SPC25 70–224 is also unable to bind MIS12C NSL1-258EE , indicating that the binding sites for HP1-α and SPC24 57–197 –SPC25 70–224 overlap, at least in part (middle). When HP1-α and SPC24 57–197 –SPC25 70–224 were combined in stoichiometric amounts with MIS12C NSL1-258 , HP1-α did not coelute with MIS12C NSL1-258 , whereas SPC24 57–197 –SPC25 70–224 was incorporated in a complex with MIS12C NSL1-258 , suggesting that SPC24 57–197 –SPC25 70–224 binds MIS12C NSL1-258 with higher affinity (bottom). (A and B) Dashed gray lines and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively. (C) ITC binding curve for the interaction of MIS12C NSL1-258 with HP1-α. (D) Elution profile from a size-exclusion chromatography Superdex 75 PC 3.2/30 column of a fluorescein-labeled synthetic peptide encompassing residues 205–217 of NSL1 (fluorescein-NSL1 205–217 ). The red trace reports absorbance (Abs) at 525 nm. Panels D through H were run under the same experimental conditions. (E) Elution profile of the chromoshadow domain of HP1-α. (F) Elution profile of a mixture of the chromoshadow domain of HP1-α and fluorescein-NSL1 205–217 demonstrating a shift in the elution profile of the fluorescein-NSL1 205–217 peptide. (G) Elution profile of SPC24 57–197 –SPC25 70–224 . (H) The SPC24 57–197 –SPC25 70–224 construct does not coelute with the fluorescein-NSL1 205–217 peptide, suggesting that this region of NSL1 is insufficient for high-affinity binding to NDC80C. (I) Summary of interactions presented in the figure. The position of the second binding site for SPC24–SPC24, indicated by a black curved segment, is actually on MIS12C, but not necessarily on the NSL1 subunit as shown. CD, chromodomain; CSD, chromoshadow domain.

    Journal: The Journal of Cell Biology

    Article Title: The MIS12 complex is a protein interaction hub for outer kinetochore assembly

    doi: 10.1083/jcb.201002070

    Figure Lengend Snippet: Interaction of MIS12C with HP1. (A) Size-exclusion chromatography elution profiles and SDS-PAGE analysis of MIS12C NSL1-258 (top), HP1-α (middle), and their stoichiometric combination (bottom). (B) The MIS12C NSL1-258EE mutant does not bind HP1-α (top). SPC24 57–197 –SPC25 70–224 is also unable to bind MIS12C NSL1-258EE , indicating that the binding sites for HP1-α and SPC24 57–197 –SPC25 70–224 overlap, at least in part (middle). When HP1-α and SPC24 57–197 –SPC25 70–224 were combined in stoichiometric amounts with MIS12C NSL1-258 , HP1-α did not coelute with MIS12C NSL1-258 , whereas SPC24 57–197 –SPC25 70–224 was incorporated in a complex with MIS12C NSL1-258 , suggesting that SPC24 57–197 –SPC25 70–224 binds MIS12C NSL1-258 with higher affinity (bottom). (A and B) Dashed gray lines and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively. (C) ITC binding curve for the interaction of MIS12C NSL1-258 with HP1-α. (D) Elution profile from a size-exclusion chromatography Superdex 75 PC 3.2/30 column of a fluorescein-labeled synthetic peptide encompassing residues 205–217 of NSL1 (fluorescein-NSL1 205–217 ). The red trace reports absorbance (Abs) at 525 nm. Panels D through H were run under the same experimental conditions. (E) Elution profile of the chromoshadow domain of HP1-α. (F) Elution profile of a mixture of the chromoshadow domain of HP1-α and fluorescein-NSL1 205–217 demonstrating a shift in the elution profile of the fluorescein-NSL1 205–217 peptide. (G) Elution profile of SPC24 57–197 –SPC25 70–224 . (H) The SPC24 57–197 –SPC25 70–224 construct does not coelute with the fluorescein-NSL1 205–217 peptide, suggesting that this region of NSL1 is insufficient for high-affinity binding to NDC80C. (I) Summary of interactions presented in the figure. The position of the second binding site for SPC24–SPC24, indicated by a black curved segment, is actually on MIS12C, but not necessarily on the NSL1 subunit as shown. CD, chromodomain; CSD, chromoshadow domain.

    Article Snippet: Fixed concentrations (10 nM) of fluorescein-labeled NSL1 peptides (synthesized by Mimotopes) were mixed with increasing concentrations of the indicated SPC24–SPC25 or HP1 constructs in PBS buffer, and reaction mixtures were allowed to equilibrate overnight at 4°C.

    Techniques: Size-exclusion Chromatography, SDS Page, Mutagenesis, Binding Assay, Labeling, Construct

    Reconstitution and structural analysis of human MIS12C. (A) Schematic representation of the components of the MIS12C, NDC80C, and KNL1C. Coiled-coil (CC) predictions calculated with program COILS ( Lupas et al., 1991 ) are shown exclusively for subunits with partial or complete coiled-coil content. Alternate names in humans are indicated. Hs, Homo sapiens . (B) Size-exclusion chromatography run of recombinant MIS12C NSL1-258 with corresponding SDS-PAGE separation stained with Coomassie brilliant blue. The molecular mass of the recombinant complex is ∼120 kD, but the protein elutes earlier than expected for a globular protein of equivalent molecular mass, suggesting that it is an oligomer or that it is elongated. The dashed gray line and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively. a.u., arbitrary unit. (C) Negative-stain EM was performed on the recombinant MIS12C NSL1-258 . The maximum length of the complex varies between ∼21 and 23 nm depending on the curvature. The maximum thickness of the rodlike structures is ∼3 nm. (D) The class averages represent the characteristic views of the MIS12C and reveal a varying amount of curvature. Bars: (C) 10 nm; (D) 5 nm.

    Journal: The Journal of Cell Biology

    Article Title: The MIS12 complex is a protein interaction hub for outer kinetochore assembly

    doi: 10.1083/jcb.201002070

    Figure Lengend Snippet: Reconstitution and structural analysis of human MIS12C. (A) Schematic representation of the components of the MIS12C, NDC80C, and KNL1C. Coiled-coil (CC) predictions calculated with program COILS ( Lupas et al., 1991 ) are shown exclusively for subunits with partial or complete coiled-coil content. Alternate names in humans are indicated. Hs, Homo sapiens . (B) Size-exclusion chromatography run of recombinant MIS12C NSL1-258 with corresponding SDS-PAGE separation stained with Coomassie brilliant blue. The molecular mass of the recombinant complex is ∼120 kD, but the protein elutes earlier than expected for a globular protein of equivalent molecular mass, suggesting that it is an oligomer or that it is elongated. The dashed gray line and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively. a.u., arbitrary unit. (C) Negative-stain EM was performed on the recombinant MIS12C NSL1-258 . The maximum length of the complex varies between ∼21 and 23 nm depending on the curvature. The maximum thickness of the rodlike structures is ∼3 nm. (D) The class averages represent the characteristic views of the MIS12C and reveal a varying amount of curvature. Bars: (C) 10 nm; (D) 5 nm.

    Article Snippet: Fixed concentrations (10 nM) of fluorescein-labeled NSL1 peptides (synthesized by Mimotopes) were mixed with increasing concentrations of the indicated SPC24–SPC25 or HP1 constructs in PBS buffer, and reaction mixtures were allowed to equilibrate overnight at 4°C.

    Techniques: Size-exclusion Chromatography, Recombinant, SDS Page, Staining

    Interaction of MIS12C with HP1. (A) Elution profile and SDS-PAGE analysis of a stoichiometric mixture of MIS12C–KNL1 2106–2316 and NDC80C. The elution position of individual complexes in indicated. (B) ITC analysis of the interaction of MIS12C–KNL1 2106–2316 with NDC80C. (C) SPC24–SPC25 binds the C-terminal tail of NSL1 and an additional site on MIS12 (see legend to Fig. 4 I). If the PVIHL motif is mutated, SPC24–SPC25 does not bind. If SPC24–SPC24 bound KNL1 (the red arrow indicates this interaction is hypothetical), additional binding energy may be recovered, and SPC24–SPC25 would be expected to bind even with a mutated PVIHL motif. (D) Addition of KNL1 2106–2316 to MIS12C does not rescue the lack of interaction of NDC80C to the PVIHL mutant, suggesting that SPC24–SPC25 and KNL1 2106–2316 do not interact. (A and D) Dashed gray lines and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively.

    Journal: The Journal of Cell Biology

    Article Title: The MIS12 complex is a protein interaction hub for outer kinetochore assembly

    doi: 10.1083/jcb.201002070

    Figure Lengend Snippet: Interaction of MIS12C with HP1. (A) Elution profile and SDS-PAGE analysis of a stoichiometric mixture of MIS12C–KNL1 2106–2316 and NDC80C. The elution position of individual complexes in indicated. (B) ITC analysis of the interaction of MIS12C–KNL1 2106–2316 with NDC80C. (C) SPC24–SPC25 binds the C-terminal tail of NSL1 and an additional site on MIS12 (see legend to Fig. 4 I). If the PVIHL motif is mutated, SPC24–SPC25 does not bind. If SPC24–SPC24 bound KNL1 (the red arrow indicates this interaction is hypothetical), additional binding energy may be recovered, and SPC24–SPC25 would be expected to bind even with a mutated PVIHL motif. (D) Addition of KNL1 2106–2316 to MIS12C does not rescue the lack of interaction of NDC80C to the PVIHL mutant, suggesting that SPC24–SPC25 and KNL1 2106–2316 do not interact. (A and D) Dashed gray lines and numbers indicate elution markers in the size-exclusion chromatography experiments and their molecular masses (in kilodaltons), respectively.

    Article Snippet: Fixed concentrations (10 nM) of fluorescein-labeled NSL1 peptides (synthesized by Mimotopes) were mixed with increasing concentrations of the indicated SPC24–SPC25 or HP1 constructs in PBS buffer, and reaction mixtures were allowed to equilibrate overnight at 4°C.

    Techniques: SDS Page, Binding Assay, Mutagenesis, Size-exclusion Chromatography