Figure S2. Mutations on the conserved... | Rockefeller University Press

Figure S2.

$Mutations on the conserved N-terminal SP residues of HJURP do not affect CENP-C and CENP-A binding in vitro.(a) Schematic representation of HJURP truncations used to identify the CENP-C interacting region in b. (b) Fragments of HJURP C-terminal to amino acid residue 201 do not bind CENP-C in interphase extracts. Interphase extract was supplemented with specified FLAG-HJURP truncations. Coimmunoprecipitation of endogenous CENP-C was assayed by anti–CENP-C immunoblotting following immunoprecipitation (IP) of FLAG-HJURP. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. (c) Interphase or metaphase extracts were supplemented with either WT or P221A X. laevis MBP-HJURP (residues 1–300). MBP-HJURP was immunoprecipitated and submitted for mass spectrometry. Mock precipitation using whole mouse IgG served as a negative control. (d) Relative abundance of phosphopeptides from WT MBP-HJURP S220 in metaphase versus interphase egg extract. Peptide EILEK serves as a negative control. VS#PMK represents S220 phosphorylated peptide and VSPMK represents unphosphorylated peptide. (e) S220 and P221 mutations on HJURP do not affect CENP-C binding in vitro. Interaction assay was performed using in vitro translated HJURP mutant and CENP-C. Coimmunoprecipitation of CENP-C was assayed by anti–CENP-C immunoblotting following FLAG-HJURP precipitation. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. (f) Purification of recombinant Myc-CENP-A/H4 heterodimer. Coomasie stained SDS-PAGE gel of S-column fractions of purified Myc-CENP-A/H4 heterodimer. Dotted lines highlight pooled fractions. (g) Binding of CENP-A/H4 to HJURP is not affected by S220 or P221 mutations. An in vitro interaction assay was performed using the indicated in vitro translated HJURP mutant and recombinant purified Xenopus CENP-A/H4. Co-immunoprecipitation of CENP-A was assayed by anti–CENP-A immunoblotting following FLAG-HJURP precipitation. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. (h) Binding of CENP-A/H4 to HJURP is not affected by S220 or P221 mutations. The indicated in vitro translated HJURP mutant and recombinant purified Xenopus CENP-A/H4 were added to interphase egg extract. Co-immunoprecipitation of CENP-A was assayed by anti–CENP-A immunoblotting following FLAG-HJURP precipitation. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. (i) Simultaneous binding of CENP-A/H4 and CENP-C to HJURP is not affected by S220 or P221 mutations. An in vitro interaction assay was performed using the indicated in vitro translated HJURP mutant, in vitro translated CENP-C, and recombinant purified Xenopus CENP-A/H4. Coimmunoprecipitation of CENP-C and CENP-A were assayed by anti-myc immunoblotting following FLAG-HJURP precipitation. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. Source data are available for this figure: SourceData FS2.$

Mutations on the conserved N-terminal SP residues of HJURP do not affect CENP-C and CENP-A binding in vitro. (a) Schematic representation of HJURP truncations used to identify the CENP-C interacting region in b. (b) Fragments of HJURP C-terminal to amino acid residue 201 do not bind CENP-C in interphase extracts. Interphase extract was supplemented with specified FLAG-HJURP truncations. Coimmunoprecipitation of endogenous CENP-C was assayed by anti–CENP-C immunoblotting following immunoprecipitation (IP) of FLAG-HJURP. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. (c) Interphase or metaphase extracts were supplemented with either WT or P221A X. laevis MBP-HJURP (residues 1–300). MBP-HJURP was immunoprecipitated and submitted for mass spectrometry. Mock precipitation using whole mouse IgG served as a negative control. (d) Relative abundance of phosphopeptides from WT MBP-HJURP S220 in metaphase versus interphase egg extract. Peptide EILEK serves as a negative control. VS#PMK represents S220 phosphorylated peptide and VSPMK represents unphosphorylated peptide. (e) S220 and P221 mutations on HJURP do not affect CENP-C binding in vitro. Interaction assay was performed using in vitro translated HJURP mutant and CENP-C. Coimmunoprecipitation of CENP-C was assayed by anti–CENP-C immunoblotting following FLAG-HJURP precipitation. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. (f) Purification of recombinant Myc-CENP-A/H4 heterodimer. Coomasie stained SDS-PAGE gel of S-column fractions of purified Myc-CENP-A/H4 heterodimer. Dotted lines highlight pooled fractions. (g) Binding of CENP-A/H4 to HJURP is not affected by S220 or P221 mutations. An in vitro interaction assay was performed using the indicated in vitro translated HJURP mutant and recombinant purified Xenopus CENP-A/H4. Co-immunoprecipitation of CENP-A was assayed by anti–CENP-A immunoblotting following FLAG-HJURP precipitation. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. (h) Binding of CENP-A/H4 to HJURP is not affected by S220 or P221 mutations. The indicated in vitro translated HJURP mutant and recombinant purified Xenopus CENP-A/H4 were added to interphase egg extract. Co-immunoprecipitation of CENP-A was assayed by anti–CENP-A immunoblotting following FLAG-HJURP precipitation. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. (i) Simultaneous binding of CENP-A/H4 and CENP-C to HJURP is not affected by S220 or P221 mutations. An in vitro interaction assay was performed using the indicated in vitro translated HJURP mutant, in vitro translated CENP-C, and recombinant purified Xenopus CENP-A/H4. Coimmunoprecipitation of CENP-C and CENP-A were assayed by anti-myc immunoblotting following FLAG-HJURP precipitation. Mock precipitations using scrambled DNA translated in vitro (-IVT) or whole mouse IgG served as negative controls. Source data are available for this figure: SourceData FS2.

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