rabbit anti-sgol2  (Bethyl)

Journal: Current biology : CB

Article Title: Age-dependent loss of cohesion protection in human oocytes.

doi: 10.1016/j.cub.2023.11.061

Figure Lengend Snippet: Figure 2. Loss of SGO2 at the pericentromeric bridge with increased inter-sister kinetochore distance (A) Schematic summarizing SGO2 localization in metaphase I oocytes from younger women. (B) Inter-sister kinetochore distance (white dashed arrows) was determined on metaphase I chromosomes and related to the presence of the SGO2 bridge.

Article Snippet: Cells were incubated in human polyclonal anti-centromere protein antibody (1:50; Antibodies Incorporated, 15-235), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse monoclonal antiBUB1 antibody (1:100; ThermoFisher Scientific, MA15755), mouse monoclonal anti-tubulin antibody (1:200; T6074, Sigma) and guinea pig polyclonal anti-CENP-C antibody (1:200; MBL, PB030) 3% BSA in PBST at 4 C overnight.

Techniques:

Journal: Current biology : CB

Article Title: Age-dependent loss of cohesion protection in human oocytes.

doi: 10.1016/j.cub.2023.11.061

Figure Lengend Snippet: Figure 3. Loss of cohesion in metaphase II oocytes with age and absence of bridge SGO2 (A) Representative image showing presence of single chromatids (arrows) in human metaphase II oocytes from an older (age 40) woman, compared with a younger (age 30) woman without single chromatids. White boxes with dashed lines indicate examples of paired and single chromosomes in oocyte 2. SGO2 (green), inner kinetochores (CENP-C; magenta), microtubules (a-tubulin; orange), and chromosomes (Hoechst; blue) are shown. (B) Increase of single chromatids in metaphase II oocytes with maternal age. The number of single chromatids was scored relative to woman’s age. Data were fit to a sigmoidal, 4 parameter logistic curve (R2 = 0.57). Oocytes used in representative images are labeled in the graphs. (C) Increased number of single chromatids in metaphase II oocytes with an increased fraction of sister chromatid pairs lacking a SGO2 bridge. Data were fit to a sigmoidal, 4 parameter logistic curve (R2 = 0.60). (D) The relative intensity of the centromeric pool of SGO2 between paired and single chromatids was measured only from oocytes that had single chromatids. SGO2 intensity was measured in arbitrary units (a.u.) relative to the kinetochore markers CENP-C (p = 0.96, Mann-Whitney test) and CREST (p = 0.24, Mann- Whitney test). Plots show median (dashed black line) and 25th and 75th percentiles (dotted black lines). p values were calculated using the Mann-Whitney test. n.s., not significant. See also Figure S3.

Article Snippet: Cells were incubated in human polyclonal anti-centromere protein antibody (1:50; Antibodies Incorporated, 15-235), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse monoclonal antiBUB1 antibody (1:100; ThermoFisher Scientific, MA15755), mouse monoclonal anti-tubulin antibody (1:200; T6074, Sigma) and guinea pig polyclonal anti-CENP-C antibody (1:200; MBL, PB030) 3% BSA in PBST at 4 C overnight.

Techniques: Labeling, MANN-WHITNEY

Journal: Current biology : CB

Article Title: Age-dependent loss of cohesion protection in human oocytes.

doi: 10.1016/j.cub.2023.11.061

Figure Lengend Snippet: Figure 4. Co-localization of PP2A and cohesin with the SGO2 pericentromere bridge on human metaphase II chromosomes (A and B) Chromosome spreads of metaphase II-arrested human oocytes were stained with antibodies against SGO2 (green), inner kinetochores (CENP-C, magenta), and cohesin (REC8, orange). Chromosomes were stained with Hoechst (blue). (A) Representative images with white dashed line boxes indicating representative chromosome figures shown at higher magnification below. (B) Localization of REC8 at the pericentromere bridge was scored for sister chromatid pairs with and without a SGO2 bridge. Schematic representations of the data are shown below. Statistical analyses were performed using the chi-squared test (****p < 0.0001). (C and D) Chromosome spreads of metaphase II-arrested human oocytes were stained with antibodies against SGO2 (green), inner kinetochores (CENP-C, magenta), and PP2A (orange). Chromosomes were stained with Hoechst (blue).

Article Snippet: Cells were incubated in human polyclonal anti-centromere protein antibody (1:50; Antibodies Incorporated, 15-235), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse monoclonal antiBUB1 antibody (1:100; ThermoFisher Scientific, MA15755), mouse monoclonal anti-tubulin antibody (1:200; T6074, Sigma) and guinea pig polyclonal anti-CENP-C antibody (1:200; MBL, PB030) 3% BSA in PBST at 4 C overnight.

Techniques: Staining

Journal: Current biology : CB

Article Title: Age-dependent loss of cohesion protection in human oocytes.

doi: 10.1016/j.cub.2023.11.061

Figure Lengend Snippet: Figure 6. Dependence of SGO2 localization on BUB1 activity (A) Scheme of the experiment. Metaphase II oocytes from women aged % 36 years were treated with 10 mM BAY-320 (to inhibit BUB147) or DMSO (control) overnight and fixed. (B) Representative images of control and Bay-320-treated metaphase II oocytes after immunostaining with antibodies against SGO2 (green), CENP-C (inner kinetochores, magenta), and a-tubulin (microtubules, orange). White boxes with dashed lines indicate chromosomes that have been further magnified below. (C) The percentage of chromatids per oocyte with SGO2 localization at the pericentromeric bridge was scored in control and BAY-320-treated metaphase II oocytes (****p < 0.0001, Mann-Whitney test). (D) The relative intensity of the centromeric pool of SGO2 in metaphase II oocytes from control and BAY-320-treated oocytes were measured relative to CENP-C. (****p < 0.0001, Mann-Whitney test.) Plots show median (dashed black line) and 25th and 75th percentiles (dotted black lines). (E) Schematic summarizing a model for MPS1 and BUB1 in SGO2 localization in metaphase II oocytes from younger women. See also Figure S6.

Article Snippet: Cells were incubated in human polyclonal anti-centromere protein antibody (1:50; Antibodies Incorporated, 15-235), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse monoclonal antiBUB1 antibody (1:100; ThermoFisher Scientific, MA15755), mouse monoclonal anti-tubulin antibody (1:200; T6074, Sigma) and guinea pig polyclonal anti-CENP-C antibody (1:200; MBL, PB030) 3% BSA in PBST at 4 C overnight.

Techniques: Activity Assay, Control, Immunostaining, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: The Ectopic Expression of Meiosis Regulatory Genes in Cutaneous T-Cell Lymphomas (CTCL)

doi: 10.3389/fonc.2019.00429

Figure Lengend Snippet: RNA-Seq gene expression of meiCT genes (A) STRA8 , (B) STAG3 , (C) SGO2 , (D) SYCP3 , and (E) DMC1 in Sézary Syndrome patients compared to normal/control subjects based on pooled data from Choi et al. and Ungewickell et al. datasets. Data was normalized to a mean TPM value for each cohort. Asterisks indicate statistical significance.

Article Snippet: Immunohistochemistry staining was performed on FFPE tissue sections using the Leica BondTM system and the standard protocol F. Sectioned slides were stained with the following anti-human antibodies: rabbit anti- STRA8 (Novus Biologicals), rabbit anti- STAG3 (Proteintech), rabbit anti- DMC1 (Proteintech), rabbit anti- SGO2 (Novus Biologicals), and rabbit anti- SYCP3 (Abcam) using either heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1 ER1) for 20 min or with an EDTA buffer (pH 9, epitope retrieval solution 2 ER2) for 20 min.

Techniques: RNA Sequencing, Gene Expression, Control

Journal: Frontiers in Oncology

Article Title: The Ectopic Expression of Meiosis Regulatory Genes in Cutaneous T-Cell Lymphomas (CTCL)

doi: 10.3389/fonc.2019.00429

Figure Lengend Snippet: RNA-sequencing results for differentially expressed meiCT genes in two pooled independent cohorts of Sézary Syndrome patients ( 12 , 16 ).

Article Snippet: Immunohistochemistry staining was performed on FFPE tissue sections using the Leica BondTM system and the standard protocol F. Sectioned slides were stained with the following anti-human antibodies: rabbit anti- STRA8 (Novus Biologicals), rabbit anti- STAG3 (Proteintech), rabbit anti- DMC1 (Proteintech), rabbit anti- SGO2 (Novus Biologicals), and rabbit anti- SYCP3 (Abcam) using either heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1 ER1) for 20 min or with an EDTA buffer (pH 9, epitope retrieval solution 2 ER2) for 20 min.

Techniques:

Journal: Frontiers in Oncology

Article Title: The Ectopic Expression of Meiosis Regulatory Genes in Cutaneous T-Cell Lymphomas (CTCL)

doi: 10.3389/fonc.2019.00429

Figure Lengend Snippet: Results of MeiCT gene expression in lesional skin biopsy samples of patients with CTCL.

Article Snippet: Immunohistochemistry staining was performed on FFPE tissue sections using the Leica BondTM system and the standard protocol F. Sectioned slides were stained with the following anti-human antibodies: rabbit anti- STRA8 (Novus Biologicals), rabbit anti- STAG3 (Proteintech), rabbit anti- DMC1 (Proteintech), rabbit anti- SGO2 (Novus Biologicals), and rabbit anti- SYCP3 (Abcam) using either heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1 ER1) for 20 min or with an EDTA buffer (pH 9, epitope retrieval solution 2 ER2) for 20 min.

Techniques: Gene Expression, Expressing, Staining

Journal: Frontiers in Oncology

Article Title: The Ectopic Expression of Meiosis Regulatory Genes in Cutaneous T-Cell Lymphomas (CTCL)

doi: 10.3389/fonc.2019.00429

Figure Lengend Snippet: Immunohistochemistry staining of SGO2 in (A) normal human testis (positive control), (B) normal skin (C) stage IIA MF lesional skin, (D) CD8 + MF lesional skin, (E) Sézary Syndrome ( F) peripheral T-Cell Lymphoma. Scale bars are 50 μm. Nuclear staining in malignant lymphocytes is highlighted (red arrow).

Article Snippet: Immunohistochemistry staining was performed on FFPE tissue sections using the Leica BondTM system and the standard protocol F. Sectioned slides were stained with the following anti-human antibodies: rabbit anti- STRA8 (Novus Biologicals), rabbit anti- STAG3 (Proteintech), rabbit anti- DMC1 (Proteintech), rabbit anti- SGO2 (Novus Biologicals), and rabbit anti- SYCP3 (Abcam) using either heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1 ER1) for 20 min or with an EDTA buffer (pH 9, epitope retrieval solution 2 ER2) for 20 min.

Techniques: Immunohistochemistry, Staining, Positive Control

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) Silver stain of Tandem Affinity Purification (TAP) of SET-complexes from iMEFs. CYTO: cytosol, SNE: soluble nuclear extract, CHR: chromatin pellet, NFH-SET: N-terminal FLAG-HA-SET, ENDO-SET: endogenous SET. (B) Venn diagram depicting top proteins identified from TAP-MS analysis of cytosolic and nuclear extracts prepared from mouse (iMEFs) and human (hTERT-RPE1) cells. Raw peptide count numbers are presented in Table S1. Only proteins with a cumulative peptide count of 10 or higher were included in the Venn diagram. (C) Volcano plot of quantitative-MS analysis of SET-associated proteins from mitotic extracts. SGOL1 and SGOL2 are highlighted in ellipses. Note that the plot represents proteins enriched in cytosolic and nuclear fractions combined. Histones enriched in the nuclear extracts are depicted separately in Figure 1H. (D) IF analysis on human chromosomes from WT RPE1 cells depicting co-localization of SET and SGOL2 at centromeres. Chromosomal arm staining is also observed for SET. Also see Figure S1E for another zoomed in chromosome. Scale bar =5 µm. (E) Glycerol gradient analysis of SET-complexes indicating co-elution of SET and SGOL2 at fraction 7, independent of PP2A (fraction 3). Traces of peak normalized intensities for each fraction is shown below. Also see Figure S1E for the same analysis with an alternate PP2A-B subunit. (F) (Top) Domain organization and constructs of SGOL2 and SET used in the pull-down assay. D-box, KEN-box: putative APC/C recognition domains, SGO: SGO domain. The asterisks in SET DIM represent point mutations that impaired dimerization. (Bottom) In vitro pull-down assay between GST-tagged N-terminal (1–650 a.a.) or C-terminal (651–1265 a.a.) regions of SGOL2 with SET, either WT or mutant in its dimerization domain (DIM) or in its acidic tail (ACID), indicating direct association of SET with the N-terminal region of SGOL2. Dimerization, but not the acidic tail, is required for SET binding. REP1 and REP2: experimental replicates. (G) Glycerol gradient separation of H1.2 complexes indicating co-elution with SET at fractions 7–9. Traces of peak normalized intensities for each fraction is shown below. Fraction 23 was omitted for clarity. (See also Figure S1I). (H) Quantitative MS data for histones co-purifying with SET in mitotic extracts. Linker histones are the most abundant histones associated with SET in the nuclear extracts. (See also Figure S1).

Article Snippet: Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Silver Staining, Affinity Purification, Staining, Co-Elution Assay, Construct, Pull Down Assay, In Vitro, Mutagenesis, Binding Assay

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) IF analysis on WT RPE1 cells highlighting SET localization during the cell-cycle. Nuclear SET increases dramatically during G2-prophase, followed by its cytosolic presence at metaphase. Centrosomal/spindle pole localization is observed at all stages as depicted by co-staining with the centrosomal marker, γ-tubulin. Scale bar = 5 µm. (B) (Top) Schematic to study SET-SGOL2 complexes in FLAG-HA-SET expressing RPE1 cells, at different mitotic stages: Nocodazole (prometaphase arrest), BI2536 (PLK1 inhibitor; prophase and prometaphase arrest). (Bottom) FLAG-SET IP from cells treated with nocodazole (NOCO) and BI2536 indicating complex formation with SGOL2 in the cytosol (NOCO and BI2536) and nuclear fractions (BI2536-nucleus/chromosome). Quantification of the same is shown on the right. (C) (Left) Schematic to synchronize cells at prophase by double-thymidine block and release. (Right) FLAG-SET IP from prophase synchronized cells indicating SGOL2 complex formation predominantly in the nucleus (D) (Left) Schematic to synchronize cells at prometaphase by double-thymidine block and release into nocodazole. Cells were harvested by mitotic shake-off (Right) FLAG-SET IP showing that the SET-SGOL2 complex is largely cytosolic at this stage. (E) (Left) Schematic to study SET-H1 complexes using Barasertib and BI2536 (AURKB inhibitor; prophase and prometaphase arrest). (Right) FLAG-SET IP showing the enrichment of H1 complexes in the nucleus. (F) (Left) Schematic to study cell-cycle dependency of SET-H1 interaction using Roscovitine (CDK inhibitor; G1/S and G2/M arrest). (Right) FLAG H1.2 IP from RPE1 cells treated with Roscovitine show ablation of SET association with H1, but not of PAF1/LEO1, highlighting that progression through the cell-cycle is necessary for SET-H1 interaction. All complexes in (B), (C), (D), (E) and (F) were natively eluted using FLAG peptide. (See also Figure S2).

Article Snippet: Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Staining, Marker, Expressing, Blocking Assay

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) Schematic of CHREA assay to study mitotic protein eviction from chromosomal clusters. (B) (Top) Western blot of evicted proteins reveals that addition of SET, but not BSA, or NAP1 (Figure S3), results in a dose-dependent eviction of SGOL1 and SGOL2 from chromosomes. Additional controls are shown in Figure S3A. Histones H3 and H4 are not evicted. Immunoblot for H3 from the ensuing pellets of the reaction serves as loading control to gauge chromosomal input across samples. (Bottom) H3-pellet and peak normalized intensities from left. (C) (Top) Chaperone assay with SET mutants indicating that both dimerization and acidic tail domains are required for chaperone activity. (Bottom) H3-pellet and peak normalized intensities from left. (D) (Top) NFH-SET RPE1 cells treated with increasing concentrations of BI2536 (20, 100 and 500 nM) display a dose-dependent retention of SET-SGOL2, but not SET-PP2A complexes in the nucleus. (Bottom) Quantification of SET-normalized intensities. Two lines reflect replicates of the experiment. See also Figure S3D for total levels of SGOL2 and SET under these conditions. (E) Schematic of the two-step CHREA in the presence of PLK1. (F) Results of a kinase assay [step 1 of (E)] visualized by autoradiography along with the corresponding Coomassie Blue staining. PLK1 phosphorylates chromosomal clusters used in the chaperone assay. (G) (Left) CHREA performed as in step 2 of (E) indicating that SET evicts SGOL1 and SGOL2 from PLK1 pre-phosphorylated chromosomal clusters, with higher efficiency than those phosphorylated with PLK1+BI2536. (Right) H3-pellet and peak normalized intensities from top. (H) PLK1 kinase assay with purified N-terminal and C-terminal domains of SGOL2. The C-terminal region of SGOL2 is preferentially phosphorylated by PLK1. (See also Figure S3).

Article Snippet: Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Western Blot, Activity Assay, Kinase Assay, Autoradiography, Staining, Purification

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) (Top) Giemsa staining of metaphase chromosomes showing unresolved sister-chromatids upon SET KO. Scale bar = 10 µm. (Bottom) Inter-chromatid distances are significantly reduced and percentage of metaphases with resolution defects are markedly increased. (B) FLAG-SET IP from BI2536-treated cells showing specific association with H1S/T18ph but not with other mitotic marks, H1S27ph or H1S25ph. Also see Figure S6C. CYTO: Cytosol; NUC: Nuclear Extract. (C) (Left) Giemsa staining of metaphase chromosomes isolated from cells overexpressing H1E_WT or H1E_T18A showing defective chromatid resolution in the latter case. Quantification is shown on right. (D) (Left) Dynamics of H1S/T18ph through mitosis was monitored by IF analysis. Images were captured on the same frame for comparing the different stages. Scale bar = 20 µm in the low magnification image and 10 µm on the zoomed panels. (Right) Quantification of H1S/T18ph expression by measurement of corrected total cell fluorescence. (E) Schematic to study H1 eviction from chromosomes upon SET KO. (F) Western blot analysis of chromosomes from control and SET KO cells depicting a dramatic increase in chromosomal H1S/T18ph upon SET KO. A modest increase in SGOL2 was also observed. Levels of other chromosomal proteins are largely unaltered. (G) IF analysis on metaphase chromosomes showing that H1S/T18ph is not evicted from the inter-chromatid axis upon SET KO. ACA: Anti centromere antigen. Scale bar = 10 µm. Quantification is shown on the right. The staining intensity with H1S/T18ph antibody was weak and the green channel was enhanced equally for all control and SET KO images. (H) Chromosome IF analysis of SET (Left) and H1S/T18ph (Right) in RPE1 WT cells depicting strong inter-chromatid axial staining. In addition, SET also localizes to centromeres. Scale bar = 10 µm. For quantification panels in 5A, 5C, 5D and 5G: N numbers are indicated on the y-axis. *** = p<0.0001, Fisher’s exact test. (See also Figures S5 and S6).

Article Snippet: Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Staining, Isolation, Expressing, Fluorescence, Western Blot

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) Schematic of CHREA to study H1S/T18ph eviction. (B) (Top) Western blot analysis of evicted proteins showing eviction of H1S/T18ph (as well as SGOL2) upon rescue with WT, but not ACID or DIM SET. REP1 and REP2 are two replicates of the experiment. (Bottom) Quantification of western blot on top. (C) Schematic of CHREA for analyzing effect of phosphorylation on H1 eviction by treatment with λ phosphatase (PPASE) or λ phosphatase with phosphatase inhibitors (PPASE+ Inhibitors). (D) (Right) Western blot analysis of evicted proteins showing a significant reduction in H1 eviction upon phosphatase treatment. Note that recognition by the H1S/T18ph mark is lost upon phosphatase treatment. (Left) Quantification of evicted proteins. (E) Model depicting the function of SET as a mitotic chaperone. See discussion for description.

Article Snippet: Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Western Blot

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) (Left) Schematic of CRISPR-Cas9 mediated SET knockout (SET KO) in RPE1 cells. (Right) Western blot of whole-cell lysate confirming the knockout. (B) Growth curve of control and SET KO cells indicating severe growth-impairment upon SET KO. Number of replicates = 3 for each time point. *** = p <0.0001, two-way ANOVA test. (C) (Left) Panels from live-cell imaging of control and SET KO cells expressing H2B-RFP. Mitoses marked with asterisks are zoomed on the insets. Scale bar = 10 µm. SET KO cells display a significant delay in prophase-metaphase transition and increased frequencies of abnormal mitoses (quantification shown on right), including the presence of lagging chromosomes (arrowheads on SET KO panel- 80 min) and occurrence of micronuclei (arrows on SET KO panels −100 min and 110 min). (D) IF analysis at various mitotic stages showing that SET KO results in SGOL2 and SGOL1 (Figure S4C), persistence beyond anaphase into telophase and interphase. Scale bar = 5 µm. Quantification is shown on the right. For all quantification panels in (C) and (D): ***=p<0.0001, Fisher’s exact test, N numbers are indicated on the y-axis. (See also Figure S4, movies S1 and S2).

Article Snippet: Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: CRISPR, Knock-Out, Western Blot, Live Cell Imaging, Expressing