Journal: bioRxiv

Article Title: Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

doi: 10.1101/2021.11.02.466959

Figure Lengend Snippet: a. ICD components distribution during metaphase-I spermatocytes. Double immunolabelling of (A-B) H3T3ph (green) and SYCP3 (red), (C-D) AURKph (green) and SYCP3 (red), (E-F) H2AT120ph (green) and SYCP3 (red), and (G-H) SGO2 (green) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Scale bar in H represents 10 µm. b. ICD components distribution during metaphase-II spermatocytes. Double immunolabelling of (A-B) H3T3ph (green) and SYCP3 (red), (C-D) AURKph (green) and SYCP3 (red), (E-F) H2AT120ph (green) and SYCP3 (red), and (G-H) SGO2 (green) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Scale bar in H represents 10 µm. c. Quantitative analysis of ICD components signals in metaphase-I spermatocytes. Experiments were conducted for one biological replicates. Data represents mean ± SEM, **** p < 0.0001, Student’s t -test. d. Quantitative analysis of ICD components signals in metaphase-II spermatocytes. Experiments were conducted for one biological replicates. Data represents mean ± SEM, **** p < 0.0001, Student’s t -test.

Article Snippet: Histone modifications were detected with the following primary antibodies: rabbit polyclonal anti-H2AT120ph antibody (Active Motif, 39391) at a 1:10 dilution, rabbit polyclonal anti-H3T3ph antibodies (Abcam, ab-17532; and Upstate, 07-424) at a 1:800 dilution.

Techniques: Staining

Journal: bioRxiv

Article Title: Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

doi: 10.1101/2021.11.02.466959

Figure Lengend Snippet: a. ICD components distribution during metaphase-I. Double immunolabelling of (A-B) H3T3ph (green) and SYCP3 (red), (C-D) AURKph (green) and SYCP3 (red), (E-F) H2AT120ph (green) and SYCP3 (red), and (G-H) SGO2 (green) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Scale bar in H represents 10 µm. b. ICD components distribution during metaphase-II spermatocytes. Double immunolabelling of (A-B) H3T3ph (green) and SYCP3 (red), (C-D) AURKph (green) and SYCP3 (red), (E-F) H2AT120ph (green) and SYCP3 (red), and (G-H) SGO2 (green) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Scale bar in H represents 10 µm. c. Quantitative analysis of ICD components signals in metaphase-I spermatocytes. Experiments were conducted for two biological replicates. Data represents mean ± SEM, **** p < 0.0001, Student’s t -test. d. Quantitative analysis of ICD components signals in metaphase-II spermatocytes. Experiments were conducted for two biological replicates. Data represents mean ± SEM, **** p < 0.0001, Student’s t -test.

Article Snippet: Histone modifications were detected with the following primary antibodies: rabbit polyclonal anti-H2AT120ph antibody (Active Motif, 39391) at a 1:10 dilution, rabbit polyclonal anti-H3T3ph antibodies (Abcam, ab-17532; and Upstate, 07-424) at a 1:800 dilution.

Techniques: Staining

Journal: Nature Communications

Article Title: Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch

doi: 10.1038/ncomms10215

Figure Lengend Snippet: ( a ) Sequence alignment of the PP1-interaction domains of RepoMan, PNUTS and Spinophilin. ( b ) Conservation of the PP1-interaction domain of RepoMan in vertebrates. ( c ) EGFP traps from HEK293T cells expressing the indicated EGFP-RepoMan mutants were analysed by immunoblotting for the binding of PP1. ( d ) U2OS cells were transfected with EGFP-tagged RepoMan-WT or RepoMan-3A. EGFP traps from non-synchronized (Nonsync) or Nocodazole-arrested (Mitosis) U2OS cell lysates were examined for associated PP1. ( e ) U2OS cells were transfected with EGFP-tagged RepoMan-WT. EGFP traps from Nonsync or Nocodazole-arrested (Mitosis) U2OS cell lysates were examined for EGFP and RepoMan-T412ph antibody. ( f ) EGFP traps from non-synchronized HEK293T cells expressing EGFP-tagged β-galactosidase (β-gal), RepoMan-WT, RepoMan-T412D or RepoMan-3D were analysed by immunoblotting for associated PP1. ( g ) The PP1/EGFP ratio was quantified by densitometric analysis. The data represent means±s.e.m. from three independent experiments. ( h ) EGFP traps from non-synchronized HEK293T cells expressing EGFP-tagged RepoMan-WT or RepoMan-3A were phosphorylated in vitro with recombinant Cdk2/Cyclin A. Unbound PP1 was washed away before the addition of SDS sample buffer to the traps and immunoblotting for PP1. ( i ) Phosphorylation of the C-terminal Cdk site of PP1 (PP1ph) was examined by immunoblotting of EGFP traps from U2OS cells expressing EGFP-RepoMan-WT in Nocodazole-arrested (Mitosis) or non-synchronized (Nonsync) U2OS cells with a phospho-epitope specific antibody. ( j ) EGFP traps from non-synchronized HEK293T cells expressing EGFP-RepoMan-3A (RM 3A) were phosphorylated with Cdk2/Cyclin A. The unbound fractions were washed away before the addition of SDS sample buffer to the traps and immunoblotting for PP1 and PP1ph.

Article Snippet: Antibodies against EGFP (SC-8334, Santa Cruz, 1:1,000), GAPDH (2118, Cell Signaling, 1:5,000), H3S10ph (9706, Cell Signaling, 1:1,000), ACA (HCT-0100, ImmunoVision, 1:4,000), Aurora B (611082, BD Transduction Laboratories, 1:250), H3T3ph (07–424, Millipore, 1:10,000), RepoMan (HPA030049, Sigma, 1:300 for IF, 1:1,000 for WB; Ab45129, Abcam, 1:1,000 for WB), PP2A-Cα (610556, BD Transduction Laboratories, 1:2,000), PP2A-B56γ (sc-67038, Santa Cruz, 1:1,000), Lamin B1 (sc-6216, Santa Cruz, 1:500), phospho-PP1 (2581S, Cell Signaling, 1:1,000), Importin β (ab2811, Abcam, 1:10,000 for IF, 1:2,000 for WB) and Nup153 (ab84872, abcam, 1:5,00) and phospho-Ser/Thr-Pro, also known as mpm2 (Merckmillipore, 05–368, 1:1,000) were purchased.

Techniques: Sequencing, Expressing, Western Blot, Binding Assay, Transfection, In Vitro, Recombinant, Phospho-proteomics

Journal: Nature Communications

Article Title: Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch

doi: 10.1038/ncomms10215

Figure Lengend Snippet: During (pro)metaphase, Cdk1 promotes Aurora-B activation and prevents nuclear-envelope reassembly through phosphorylation of RepoMan at multiple sites, which reduces the binding of PP1 and Importin β. In contrast, the Cdk1-mediated phosphorylation of RepoMan at S591 enhances the recruitment of PP2A, which contributes to the dynamic chromosome targeting of RepoMan in (pro)metaphase and the centromeric localization of Aurora B. After anaphase onset, Cdk1 inactivation leads to the dephosphorylation of RepoMan, associated with the loss of PP2A and the bulk recruitment of PP1-RepoMan to the chromosomes. PP1-RepoMan then inactivates Aurora B and promotes nuclear-envelope reassembly through dephosphorylation of Histone H3 at T3, Aurora B at T232 and RepoMan at S893 and the Importin-β-binding domain.

Article Snippet: Antibodies against EGFP (SC-8334, Santa Cruz, 1:1,000), GAPDH (2118, Cell Signaling, 1:5,000), H3S10ph (9706, Cell Signaling, 1:1,000), ACA (HCT-0100, ImmunoVision, 1:4,000), Aurora B (611082, BD Transduction Laboratories, 1:250), H3T3ph (07–424, Millipore, 1:10,000), RepoMan (HPA030049, Sigma, 1:300 for IF, 1:1,000 for WB; Ab45129, Abcam, 1:1,000 for WB), PP2A-Cα (610556, BD Transduction Laboratories, 1:2,000), PP2A-B56γ (sc-67038, Santa Cruz, 1:1,000), Lamin B1 (sc-6216, Santa Cruz, 1:500), phospho-PP1 (2581S, Cell Signaling, 1:1,000), Importin β (ab2811, Abcam, 1:10,000 for IF, 1:2,000 for WB) and Nup153 (ab84872, abcam, 1:5,00) and phospho-Ser/Thr-Pro, also known as mpm2 (Merckmillipore, 05–368, 1:1,000) were purchased.

Techniques: Activation Assay, Phospho-proteomics, Binding Assay, De-Phosphorylation Assay