Journal: Communications Biology

Article Title: BubR1 TPR domain supports the mitotic checkpoint by promoting MCC formation and MCC-APC/C interaction

doi: 10.1038/s42003-026-10384-9

Figure Lengend Snippet: A Predicted BubR1 structure by AlphaFold . B Schemes showing structures of wild-type BubR1 and two BubR1 TPR mutants. K1: KEN1, residues 26–28; D1: D box 1, residues 224–232; A1: ABBA1, residues 272–277; K2: KEN2, residues 304–306; A2: ABBA2, residues 340–345; A3: ABBA3, residues 528–533; D2: D box 2, residues 555–563; TPR: tetratricopeptide repeat domain, residues 59–219; kinase: pseudokinase domain, residues 766–1050. TPR in orange indicates the original BubR1 TPR. TPR in red indicates the replaced Bub1 TPR. C Plot showing the time from NEBD to mitotic exit of the cells transfected with siRNA oligos against BubR1 and RNAi-resistant constructs expressing YFP-BubR1. Low dose of nocodazole (40 ng/ml) was applied into the medium before live cell imaging was conducted. Each circle represents the time spent in mitosis of a single cell. The red line indicates the median time with interquartile range. The number of cells analyzed per condition is indicated above ( n = X). Mann-Whitney U -test was applied. ns means not significant; **** means P < 0.0001. D YFP-BubR1 or YFP-BubR1 B1TPR was expressed in HeLa cells and immunoprecipitated by GFP-trap beads. YFP-BubR1, Apc4, Apc7, Cdc20 and Mad2 were detected by western blot from the immunoprecipitate. E Overview of the BubR1 mutants used in the following study for specific interaction disruption.

Article Snippet: Antibodies used in this study include YFP antibody for YFP-BubR1 detection (homemade in JN lab , 1:2000), Cdc20 (Santa Cruz, sc-13162, 1:1000), Mad2 (Bethyl, A300-301A, 1:1000), Apc7 (Santa Cruz, sc- 365649, 1:1000), Apc4 (Santa Cruz, sc-514895, 1:1000).

Techniques: Transfection, Construct, Expressing, Live Cell Imaging, Single Cell, MANN-WHITNEY, Immunoprecipitation, Western Blot, Disruption

Journal: Communications Biology

Article Title: BubR1 TPR domain supports the mitotic checkpoint by promoting MCC formation and MCC-APC/C interaction

doi: 10.1038/s42003-026-10384-9

Figure Lengend Snippet: A Structural analysis of BubR1 TPR-Mad2 interaction from PDB: 6TLJ. B Plot showing the time from NEBD to mitotic exit of the cells transfected with siRNA oligos against BubR1 and RNAi-resistant constructs expressing YFP-BubR1 E23A or W22A/E23A. Low dose of nocodazole (40 ng/ml) was applied to the medium before live cell imaging conducted. Each circle represents the time spent in mitosis of a single cell. The red line indicates the median time with interquartile range. The number of cells analyzed per condition is indicated above ( n = X). Mann-Whitney u-test was applied. **** means P < 0.0001. C YFP-BubR1 or YFP-BubR1 W22 A/E23A was expressed in HeLa cells and immunoprecipitated by GFP-trap beads. YFP-BubR1, Apc4, Apc7, Cdc20 and Mad2 were detected by western blot from the immunoprecipitate. D In vitro protein binding assay showing direct interaction between BubR1 TPR and Mad2. Left: western blot showing the presence of His-tagged Mad2 pulled down together with GST-tagged BubR1 TPR recombinant protein. Right: quantification of the Western blot results from three repeats. The line represents the mean value of three repeats with standard error of the mean. The value of GST alone was subtracted from the values of the GST-BubR1 TPR recombinant proteins and set to zero. The value from wild-type GST-BubR1 TPR was set to 1, and the value from GST-BubR1 TPR 2A was normalized to the wild-type value.

Article Snippet: Antibodies used in this study include YFP antibody for YFP-BubR1 detection (homemade in JN lab , 1:2000), Cdc20 (Santa Cruz, sc-13162, 1:1000), Mad2 (Bethyl, A300-301A, 1:1000), Apc7 (Santa Cruz, sc- 365649, 1:1000), Apc4 (Santa Cruz, sc-514895, 1:1000).

Techniques: Transfection, Construct, Expressing, Live Cell Imaging, Single Cell, MANN-WHITNEY, Immunoprecipitation, Western Blot, In Vitro, Protein Binding, Recombinant

Journal: Communications Biology

Article Title: BubR1 TPR domain supports the mitotic checkpoint by promoting MCC formation and MCC-APC/C interaction

doi: 10.1038/s42003-026-10384-9

Figure Lengend Snippet: A Structural analysis of BubR1 TPR-Apc2 interaction from PDB: 6TLJ. B Plot showing the time from NEBD to mitotic exit of the cells transfected with siRNA oligos against BubR1 and RNAi-resistant constructs expressing YFP-BubR1 or YFP-BubR1 R169A/F175A/V200A/L205A (RFVL/4 A). Low dose of nocodazole (40 ng/ml) was applied into the medium before live cell imaging conducted. Each circle represents the time spent in mitosis of a single cell. Red line indicates the median time with interquartile range. The number of cells analyzed per condition is indicated above ( n = X). Mann-Whitney U -test was applied. **** means P < 0.0001. C YFP-BubR1 or YFP-BubR1 RFVL/4 A was expressed in HeLa cells and immunoprecipitated by GFP-trap beads. YFP-BubR1, Apc4, Apc7, Cdc20 and Mad2 were detected by western blot from the immunoprecipitate. D Plot showing the time from NEBD to mitotic exit of the cells treated similarly as in ( B ). E Immunoprecipitation of YFP-BubR1 or YFP-BubR1 R169A/F175A or YFP-BubR1 V200A/L205A in cells treated similarly as in ( C ).

Article Snippet: Antibodies used in this study include YFP antibody for YFP-BubR1 detection (homemade in JN lab , 1:2000), Cdc20 (Santa Cruz, sc-13162, 1:1000), Mad2 (Bethyl, A300-301A, 1:1000), Apc7 (Santa Cruz, sc- 365649, 1:1000), Apc4 (Santa Cruz, sc-514895, 1:1000).

Techniques: Transfection, Construct, Expressing, Live Cell Imaging, Single Cell, MANN-WHITNEY, Immunoprecipitation, Western Blot

Journal: Communications Biology

Article Title: BubR1 TPR domain supports the mitotic checkpoint by promoting MCC formation and MCC-APC/C interaction

doi: 10.1038/s42003-026-10384-9

Figure Lengend Snippet: A Structural analysis of BubR1 TPR-Cdc20 APC/C interaction from PDB: 6TLJ. Insert in the middle shows residues involved in the interaction. Insert in the right shows electrostatic potential around the interaction interface. Red represents negatively charged region; blue represents positively charged region; white represents neutral region. B-C Plots showing the time from NEBD to mitotic exit of the cells transfected with siRNA oligos against BubR1 or Cdc20 and RNAi-resistant constructs expressing YFP-BubR1 or YFP-Cdc20 or corresponding mutants. A low dose of nocodazole (40 ng/ml) was applied to the medium before live cell imaging was conducted. Each circle represents the time spent in mitosis of a single cell. The red line indicates the median time with interquartile range. The number of cells analyzed per condition is indicated above ( n = X). Mann-Whitney U -test was applied. * means P < 0.1; *** means P < 0.001; **** means P < 0.0001. D YFP-BubR1 or YFP-BubR1 E134R or YFP-BubR1 D137R or YFP-BubR1 C132E expressed in HeLa cells was immunoprecipitated by GFP-trap beads and examined the presence of associated proteins, including Apc4, Apc7, Cdc20 and Mad2. E-F Plots showing the time from NEBD to mitotic exit of BubR1 mutants similarly as in ( B ). G YFP-BubR1 or YFP-BubR1 E134R/D137R or YFP-BubR1 W125A/L128A/C132A/M138A (WLCM/4 A) was expressed in HeLa cells and immunoprecipitated by GFP-trap beads similarly as in ( D ).

Article Snippet: Antibodies used in this study include YFP antibody for YFP-BubR1 detection (homemade in JN lab , 1:2000), Cdc20 (Santa Cruz, sc-13162, 1:1000), Mad2 (Bethyl, A300-301A, 1:1000), Apc7 (Santa Cruz, sc- 365649, 1:1000), Apc4 (Santa Cruz, sc-514895, 1:1000).

Techniques: Transfection, Construct, Expressing, Live Cell Imaging, Single Cell, MANN-WHITNEY, Immunoprecipitation

Journal: Communications Biology

Article Title: BubR1 TPR domain supports the mitotic checkpoint by promoting MCC formation and MCC-APC/C interaction

doi: 10.1038/s42003-026-10384-9

Figure Lengend Snippet: A Structural analysis of BubR1 intramolecular interaction from PDB: 6TLJ. B Plot showing the time from NEBD to mitotic exit of the cells transfected with siRNA oligos against BubR1 and RNAi-resistant constructs expressing YFP-BubR1 or corresponding mutants. Low dose of nocodazole (40 ng/ml) was applied to the medium before live cell imaging conducted. Each circle represents the time spent in mitosis of a single cell. Red line indicates the median time with interquartile range. The number of cells analyzed per condition is indicated above ( n = X). Mann-Whitney u- test was applied. **** means P < 0.0001. C YFP-BubR1 or YFP-BubR1 Y141A or YFP- BubR1 W295A was expressed in HeLa cells and immunoprecipitated by GFP-trap beads. YFP-BubR1, Apc4, Apc7, Cdc20 and Mad2 were detected by western blot from the immunoprecipitate. D Local sequence alignment was performed with part of BubR1 lasso region from several species, showing the conservation of the key residues required for the lasso formation. E Cartoon showing the presence of the lasso structure formed from the intramolecular binding on BubR1 of MCC complex and the full occupation of Cdc20 APC/C in APC/C complex.

Article Snippet: Antibodies used in this study include YFP antibody for YFP-BubR1 detection (homemade in JN lab , 1:2000), Cdc20 (Santa Cruz, sc-13162, 1:1000), Mad2 (Bethyl, A300-301A, 1:1000), Apc7 (Santa Cruz, sc- 365649, 1:1000), Apc4 (Santa Cruz, sc-514895, 1:1000).

Techniques: Transfection, Construct, Expressing, Live Cell Imaging, Single Cell, MANN-WHITNEY, Immunoprecipitation, Western Blot, Sequencing, Binding Assay

Journal: iScience

Article Title: hSpindly dynamics modulate spindle assembly checkpoint signaling, promoting resistance to mitotic inhibitors

doi: 10.1016/j.isci.2026.116312

Figure Lengend Snippet: hSpindly as a component of SAC machinery (A) Stable inducible RPE-1 GFP-hSpindly cells were treated or not with doxycycline (DOXY). Cell extracts were prepared and immunoblotted with the indicated antibodies. (B) Representative images of hSpindly levels at kinetochores of RPE-1 GFP-hSpindly treated or not with DOXY in the presence of 3.3 μM NZ. Endogenous or exogenous hSpindly were visualized by hSpindly antibody (in the absence of DOXY) and exogenous hSpindly was visualized by GFP (in the presence of DOXY). Bub1 or CENP-C antibodies were used as kinetochore markers, and DAPI to visualize the DNA. Scale bars, 10 μm; insets, 2× zoom. (C) Violin plots representing intensity or total area of hSpindly ENDO , hSpindly EXO and Bub1 at kinetochores from the experiment shown in (B). Lines indicate mean or standard deviation. Statistical analysis was performed with a non-parametric test comparing two unpaired groups (∗∗∗, p < 0.001; hSpindly ENDO n , 1,074; hSpindly EXO n , 979). (D) RPE-1 GFP-hSpindly cell extracts after NZ and DOXY treatment for 24 h were immunoprecipitated with anti-hSpindly (I) or normal mouse serum (PI) as control. Immunoprecipitated materials were analyzed by western blot using the antibodies indicated. (E and F) RPE-1 GFP-hSpindly cell extracts treated with NZ and DOXY for 24 h were immunoprecipitated using anti-Mad1 or anti-Mad2 antibodies (I), respectively, with normal mouse serum used as a control (PI). Western blot analysis was performed using the specified antibodies. Anti-Mad2 was used as a control of Mad1 immunoprecipitation (E) due to the poor performance of Mad1 in western blot. E, extracts; Mw, molecular weight; I, immune; PI, preimmune. The upper band marked with asterisk is an unspecific band. Histograms show the densitometric analysis of the indicated proteins. Error bars represent the SD (∗, p < 0.05, Student’s t test). Western Blot data are representative of at least two or three independent experiments.

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A ); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Standard Deviation, Immunoprecipitation, Control, Western Blot, Molecular Weight

Journal: iScience

Article Title: hSpindly dynamics modulate spindle assembly checkpoint signaling, promoting resistance to mitotic inhibitors

doi: 10.1016/j.isci.2026.116312

Figure Lengend Snippet: hSpindly overexpression maintains SAC activation by enhancing Mad2 recruitment at kinetochores (A) RPE-1 GFP-hSpindly cells were washed out after 24 h of NZ treatment without or with DOXY and collected every 30 min. Cell extracts were analyzed by western blot with the antibodies indicated (top). (B) RPE-1 GFP-hSpindly cells were treated with NZ with or without DOXY for 24 h and collected after 1 μm reversine addition at indicated times. Cell extracts were prepared and immunoblotted with the indicated antibodies (top). Bar graphs show the rate of cyclin B degradation, calculated as the slope of cyclin B reduction over the time (bottom A and B). Error bars represent the SD (∗, p < 0.05, Student’s t test). Western blot data are representative of at least three independent experiments. (C) Representative images of GFP-hSpindly, Bub1, CENP-C, or Mad2 levels at kinetochores in RPE-1 GFP-hSpindly cells treated or not with DOXY in the presence of NZ. DAPI to visualize the DNA. Scale bars, 10 μm; insets, 2× zoom. (D) Boxplots representing intensity of Mad2 at kinetochores from the experiment shown in (C). Black lines indicate mean. Statistical analysis was performed with a non-parametric test comparing two unpaired groups (∗∗∗, p < 0.001; –DOXY n , 632; +DOXY n , 887).

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A ); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Over Expression, Activation Assay, Western Blot

Journal: iScience

Article Title: hSpindly dynamics modulate spindle assembly checkpoint signaling, promoting resistance to mitotic inhibitors

doi: 10.1016/j.isci.2026.116312

Figure Lengend Snippet: Mad1-Mad2 complexes are recruited at kinetochores in the absence of the KBB pathway (A) RPE-1 GFP-hSpindly prometaphase cells induced by DOXY were treated or not with 10 μM BAY 1816032 inhibitor for 5 h in the presence of 20 μM MG132. Cell extracts were immunoprecipitated with hSpindly (I) and normal mouse serum (PI) as a control. Immunoprecipitated material was analyzed by western blot (top). (B) RPE-1 GFP-hSpindly were transfected with siRNABub1 for 48 h. Cells were treated with NZ and DOXY 24 h before harvesting. Extracts were used to immunoprecipitate Mad1 (I) using normal mouse serum as a control (PI). Complexes were analyzed by immunoblotting with antibodies as indicated (top). Histograms show the densitometric analysis of the indicated proteins. Error bars represent the SD (∗, p < 0.05, Student’s t test) (bottom of A and B). Western blot data are representative of at least two or three independent experiments. (C) Representative images of RPE-1 GFP-hSpindly prometaphase cells in the presence or not of DOXY after Bub1 or KNL1 siRNA treatment for 48 h. Scale bars, 10 μm; insets, 2× zoom. (D) Scatter dot boxes representing intensity of Mad2 at kinetochores from the experiment shown in (C). Black lines indicate mean. Statistical analysis was performed with a non-parametric test comparing two unpaired groups. (∗∗∗, p < 0.001; siRNABub1-DOXY n , 6,839; siRNABub1+DOXY n , 1,557; siRNAKNL1-DOXY n , 2,180; siRNAKNL1+DOXY n , 965). (E) Representative images of a magnification of kinetochores in RPE-1 GFP-hSpindly prometaphase cells after 48 h of Bub1 siRNA in the presence of DOXY. Scale bars, 10 μm. (F) Boxplots representing colocalization of Mad2-hSpindly at kinetochores from the experiment shown in (E). The Manders’ M 1 coefficient is shown. Statistical analysis was performed with a parametric model comparing two unpaired groups. (∗∗, p < 0.01; Mock n , 351; siRNABub1 n , 855). (G) Colony assays showing cell viability for RPE-1 GFP-hSpindly cells transfected with siRNABub1 were performed by measuring absorbance 570 nm and represented in graphs. Error bars represent the SD (∗, p < 0.05 Student’s t test). All assays were carried out in triplicate. E, extracts; Mw, molecular weight; I, immune; PI, preimmune.

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A ); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Immunoprecipitation, Control, Western Blot, Transfection, Molecular Weight

Journal: iScience

Article Title: hSpindly dynamics modulate spindle assembly checkpoint signaling, promoting resistance to mitotic inhibitors

doi: 10.1016/j.isci.2026.116312

Figure Lengend Snippet: hSpindly T552A is localized at kinetochore as wild-type and interacts with SAC proteins (A) Stable inducible RPE-1 GFP-hSpindly wild-type (wt) or GFP-hSpindly T552A (T552A) cells were treated or not with doxycycline (DOXY) in the presence of NZ for 24 h. Cell extracts were prepared and immunoblotted with the indicated antibodies. Histograms show the densitometric analysis of GFP-hSpindly or GFP-hSpindly T552A expression. Error bars represent the SD (∗∗, p < 0.01 Student’s t test). (B) Representative images of hSpindly T552A levels at kinetochores of RPE-1 GFP-hSpindlyT552A prometaphase cells treated or not with DOXY. hSpindlyT552A or Mad2 were visualized by GFP or anti-Mad2, respectively. Bub1 or CENP-C antibodies were used as kinetochore markers, and DAPI to visualize the DNA. Scale bars, 10 μm; insets, 2× zoom. (C) Violin plots representing intensity or total area of hSpindly T552A compared with hSpindly wild-type (data C). Black dots or lines indicate mean or standard deviation. Statistical analysis was performed with a non-parametric test comparing two unpaired groups. (ns, not significant; hSpindly wt n , 1,150; hSpindly T552A n , 895). (D) Distribution of hSpindly EXO or hSpindly-T552A EXO populations based on kinetochore signal intensity and area. Each dot represents a kinetochore; lines indicate the mean. (E) RPE-1 GFP-hSpindly wt or GFP-hSpindly T552A induced cell extracts after NZ treatment were immunoprecipitated with GFP-Trap beads. Immunoprecipitated materials were analyzed by western blot using the antibodies indicated. E, extracts; I, immune. Histograms show the densitometric analysis of the indicated proteins. Error bars represent the SD (∗, p < 0.05, Student’s t test). Western blot data are representative of at least two or three independent experiments.

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A ); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Expressing, Standard Deviation, Immunoprecipitation, Western Blot

Journal: iScience

Article Title: hSpindly dynamics modulate spindle assembly checkpoint signaling, promoting resistance to mitotic inhibitors

doi: 10.1016/j.isci.2026.116312

Figure Lengend Snippet: hSpindly T552A recruits Mad2 at kinetochores independently of Bub1 (A) Representative images of RPE-1 GFP-hSpindlyT552A prometaphase cells in the presence or not of DOXY after Mock or Bub1 siRNA treatment for 48 h. GFP-hSpindly T552A, Bub1, CENP-C, Mad2 were visualized with green fluorescence protein or by using the indicated antibodies. Scale bars, 10 μm; insets, 2× zoom. (B) Boxplots representing intensity of Mad2 at kinetochores from the experiment shown in (A). Black lines indicate mean. Statistical analysis was performed with a non-parametric test comparing two unpaired groups (∗∗∗, p < 0.001; Mock-DOXY n , 463; Mock+DOXY n , 88; siRNABub1-DOXY n , 50; siRNABub1+DOXY n , 46). (C) Representative images of a magnification of Kinetochores in RPE-1 GFP-hSpindly T552A prometaphase cells after 48 h of Mock or Bub1 siRNA in the presence of DOXY. Scale bars, 10 μm. (D) Boxplots representing colocalization of Mad2-hSpindly at kinetochores from the experiment shown in (C). The Manders’ M 1 coefficient is shown. Black lines indicate mean. Statistical analysis was performed with a non-parametric test comparing two unpaired groups. (ns, not significant; Mock n , 236; siRNABub1 n , 185).

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A ); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Fluorescence

Journal: iScience

Article Title: hSpindly dynamics modulate spindle assembly checkpoint signaling, promoting resistance to mitotic inhibitors

doi: 10.1016/j.isci.2026.116312

Figure Lengend Snippet: Phosphorylation of threonine 552 determines cellular resistance to anti-mitotic agents (A) Colony assays showing cell viability of RPE-1 GFP-hSpindly or RPE-1 GFP-hSpindly T552A were performed after 24 h of NZ treatment in the presence of DOXY and in the absence of both endogenous hSpindly and KNL1. Cell viability or colony forming units were quantified and represented in graphs. Error bars represent the SD (∗∗∗, p < 0.001, ∗∗, p < 0.01 Student’s t test). All assays were carried out in triplicate. (B) Model to illustrate how spindle checkpoint regulation is controlled by the KBB and RZZ pathways at unattached kinetochores. Bub1 kinase activity likely controls the activation of the RZZ pathway by hSpindly phosphorylation. hSpindly acts as a recruiter of the Mad1-Mad2 complex controlling the MCC formation with the KBB pathway.

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A ); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Phospho-proteomics, Activity Assay, Activation Assay

Journal: Science Advances

Article Title: Deubiquitinase USP8 regulates the spindle assembly checkpoint in oocytes

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) Protein levels of the MCC were assessed by immunoblots in control and USP8-depleted oocytes. The blots were probed with USP8, BUBR1, CDC20, BUB3, MAD2, and Cofilin antibodies. ( B ) Localization of BUB3 at the prometaphase I stage in control, USP8-KD, and USP8-rescue oocytes. At 3 hours after GVBD, oocytes were fixed and immunostained for BUB3, CREST, and DNA (Hoechst). Scale bar, 10 μm. ( C ) The relative fluorescence intensities of BUB3 to CREST were measured in control ( n = 200, kinetochores), USP8-KD ( n = 200, kinetochores), and USP8-rescue ( n = 200, kinetochores) oocytes. The signal intensity of BUB3 was normalized with that of CREST. ( D ) Protein levels of BUB3 were assessed by immunoblots in control, USP8-KD, and USP8-rescue oocytes. ( E ) Co-IP result showing the USP8 interaction with BUB3 in oocytes. Mouse oocytes were microinjected with USP8-Flag and BUB3-HA cRNA together or USP8-Flag cRNA alone, maintained for a further 4 hours in 200 μM IBMX to allow time for translation. Target proteins were immunoprecipitated using anti-Flag beads and subjected to Western blotting with Flag and HA antibodies. Input oocyte lysates were immunoblotted with anti-HA antibody to determine the expression of BUB3. ( F ) Co-IP result showing the BUB3 interaction with USP8 in oocytes. Mouse oocytes were microinjected with BUB3-HA and USP8-Flag cRNA together or BUB3-HA cRNA alone, maintained for a further 4 hours in 200 μM IBMX to allow time for translation. Target proteins were immunoprecipitated using anti-HA beads and subjected to Western blotting with HA and Flag antibodies. Input oocyte lysates were immunoblotted with anti-Flag antibody to determine the expression of USP8. Data were presented as the mean percentage (means ± SEM) of at least three independent experiments. *** P < 0.001.

Article Snippet: The blots were further blocked in TBST containing 5% low-fat dry milk for 1 hour at room temperature and then incubated with USP8 (1:500; Abclonal, A7031), USP8 (1:500; Proteintech, 67321-1-Ig), Cyclin B1 (1:1000; Cell Signaling Technology, 4135), BUB3 (1:1000; Abcam, ab133699), BUBR1 (1:1000; Abcam, Ab28193), MAD2 (1:1000; Proteintech, 10337-1-AP), CDC20 (1:500; Proteintech, 10252-1-AP), BUB1 (1:1000; Abcam, Ab195268 ), MPS1 (1:500; Proteintech, 10381-1-AP), Flag (1:1000; Sigma-Aldrich, F3165), HA (1:1000; Sigma-Aldrich, H9658), MYC (1:1000; Cell Signaling Technology, 2278), Normal Rabbit IgG (1:1000; Cell Signaling Technology, 2729), or Cofilin (1:5000; Proteintech, 66057-1-Ig) antibodies at 4°C overnight.

Techniques: Western Blot, Control, Fluorescence, Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Enhanced CDRs correlated with prostate cancer prognosis. (A) Visualization of core genes' dependency in different prostate cancer cells with CRISPR-Cas9 and siRNA screening. (B, C) Survival analysis of CDRs in different CRPC patient cohorts. (D) The relative expression of CDRs in normal prostate tissues and prostate cancer tissues in the TCGA-PRAD cohort. Gleason score and tumor stage correlation analysis of CDRs in different prostate cancer patient cohorts. All prostate patient cohorts were indicated in the corresponding panels. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: CRISPR, Expressing, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs linked with neuroendocrine features in prostate cancer cohorts. (A) The Venn diagram illustrates the genes specifically enhanced in prostate cancer compared with normal prostate tissues and NEPC compared with adenocarcinoma. (B, C) The relative expression of CDRs in NEPC compared with adenocarcinoma in different prostate cancer cohorts. (D) Correlation analysis of CDRs with NE score. The red indicates the higher NE score. (E, F) Pearson correlation analysis of CDRs with NE score CRPC cohort. (G) The heatmap indicates the relative expression of CDRs and AR, as well as KLK3, a transcription activity indication of AR. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NEPC, neuroendocrine prostate cancer; NE, neuroendocrine; AR, androgen receptor; KLK3, kallikrein-related peptidase 3.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, Activity Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs ablation suppressed prostate cancer cell proliferation. (A) The circuit diagram illustrates the correlation among CDRs co-expressed gene sets. (B) Venn analysis of the overlap of CDRs co-expressed genes. (C) The heatmap shows the relative expression of CDRs overlapped co-expressed genes in patients with NE signature high and low groups. (D – F) The heatmap illustrates the correlation of CDRs co-expressed genes with CDRs in ADPC (D) and CRPC (E, F) patient cohorts. (G, H) KEGG pathway analysis (G) and GSEA analysis (H) of enriched biological processes of CDRs and CDRs co-expressed genes. (I, J) The diagram illustrates the working model of CRISPR-Cas13 for RNA silencing and knockdown efficiency of CDRs with the corresponding gRNAs. (K, L) Cell growth assays indicate the impact of CDRs knockdown on the viability of different prostate cancer cells. (M) Western blotting analysis of the expression of cell cycle-regulated genes, including CCND1, CDK1, and p-CDK1, after CDRs knockdown. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). NE, neuroendocrine; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; CCND1, cyclin D1; CDK1, cyclin-dependent kinase 1.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Expressing, CRISPR, Knockdown, Western Blot, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: CDRs were transcriptionally regulated by the RB1/E2F1 axis in prostate cancer. (A) The diagrams show the canonical E2F1 motif location within the promoter of CDRs. (B) ChIP-sequencing E2F1 enrichment peak within the promoter of CDRs. (C) The relative enrichment of E2F1 within the promoter of CDRs was determined using standard ChIP-qPCR. (D) ChIP-sequencing peaks show the relative enrichment of E2F1 in CDRs' promoters after RB1 is known. (E – G) The relative expression of CDRs in CRPC patients with different RB1 deletion status. (H) CDRs expression in patients with RB1 deletion mutations from different prostate cancer cohorts. (I) qRT-PCR detected the relative expression of CDRs after RB1 or E2F1 knockdown with CRISPR-Cas13. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). RB1, retinoblastoma tumor suppressor 1; E2F1, early 2 factor 1; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time PCR.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: ChIP-sequencing, ChIP-qPCR, Expressing, Quantitative RT-PCR, Knockdown, CRISPR, Ubiquitin Proteomics, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Virtual screening identified compounds that suppressed advanced prostate cancer. (A) The diagram illustrates structure-based virtual screening strategies for CDRs-targeted compounds. (B) The heatmap shows the binding affinity of compounds with CDRs. The red indicated a higher binding affinity of compounds with the corresponding compounds. (C, D) Cell viability assays were used to determine the tumor suppressive effect of compounds with high CDRs-binding affinity in different prostate cancer cell models. (E – G) The 2D structure of Q199, XDD60, and A79, which exhibits the most significant anti-tumor efficacy in prostate cancer cell models. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2).

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: Genes & Diseases

Article Title: Integrative high-throughput studies to develop novel targets and drugs for the treatment of advanced prostate cancer

doi: 10.1016/j.gendis.2025.101732

Figure Lengend Snippet: Compounds targeting CDRs exhibited superior anti-tumor efficacy compared with AR antagonists. (A – E) The tumor cell growth inhibition effects of different dosages of Q199, XDD60, and A79, as well as enzalutamide, were determined with CCK-8 assays (A–D), and the IC 50 of each agent was calculated with three independent experiments (E). (F, G) The histograms show the relative cell viability after being treated with 5 M of Q199, XDD60, or A79 alone, or a combination. (H) The Venn diagram shows the overlap of Q199, XDD60, and A79 potential targets predicted with SwissTargetPrediction ( http://swisstargetprediction.ch/ ). Molecular docking shows the binding of CDRs with Q199, XDD60, and A79. (I) The lowest binding (LB) affinity of CDRs with Q199, XDD60, and A79. ns, not significant. ∗∗ p < 0.01. CDRs refers to CDC20 (cell division cycle 20), DTL (denticleless E3 ubiquitin protein ligase), and RRM2 (ribonucleotide reductase M2). AR, androgen receptor.

Article Snippet: Three guide RNAs (gRNA) for each target, including RB1, E2F1, CDC20, RRM2, and DTL, were designed, generated, and cloned into CRSIPR-Cas13 corresponding gRNA backbone (Addgene, 109053).

Techniques: Inhibition, CCK-8 Assay, Binding Assay, Ubiquitin Proteomics

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: K250-BubR1 acetylation is involved in the maintenance of crescent-shaped fibrous corona. ( A ) Representative images of immunostaining with anti-MAD2 and anti-ZW10 antibodies after induction of BubR1-WT, -K250R , and -K250Q expression in HeLa-FRT-TO cells. Nocodazole (200 ng/ml) alone or with MG132 was applied before fixation. Endogenous BubR1 was depleted via siRNA. Scale bar, 5 μm. ( B ) Representative SIM images of immunostained ZW10 at kinetochores of HeLa cells expressing FLAG-tagged BubR1-WT, -K250R , or -K250Q following double thymidine block and release. FRT/TO HeLa cells were treated with doxycycline for 55 h to induce expression of ectopic BubR1 variants. Endogenous BubR1 was depleted using siRNA targeting the 3′ UTR. Cells were fixed for immunostaining 10 h after the final thymidine release to enrich the mitotic population. ( C ) Effects of BubR1 acetylation status on CENP-E and RZZ complex formation. Cells were treated with nocodazole alone (+Noc) or with MG132 (+Noc + MG132). Relative protein levels were measured using a densitometer, and the ratios are indicated at the bottom. ( D ) Representative images showing immunostained ZW10 and anti-CENP-A antibodies in FRT/TO HeLa cells expressing BubR1-WT, K250R , or K250Q with (siCtrl) or without CENP-E (siCENP-E). Cells were transfected with siRNA targeting luciferase (siCtrl) or CENP-E (siCENP-E) for 48 h prior to fixation. Nocodazole was administered for 20 h before analysis.

Article Snippet: Commercially sourced antibodies used in this study include anti-BubR1 (612 503; BD Biosciences), anti-α-tubulin-FITC (F2168; Upstate), anti-Centromere protein antibody (15–235; Antibodies Inc.), anti-HDAC2 (ab12169; Abcam), anti-HDAC3 (ab7030; Abcam), anti-CDC20 (A15656; ABclonal), anti-MAD2 (A11469; ABclonal), anti-Cyclin B (sc-245; Santa Cruz Biotechnology), anti-β-actin (A700-057; Bethyl), anti-CDC27 (BD 610455; BD Biosciences), anti-FLAG (F1804; Sigma–Aldrich), anti-GAPDH (#2118; Cell Signaling Technology), anti-CENP-A (ab13939;Abcam), anti-ZW10 (sc-81430; Santa Cruz Biotechnology), anti-Myc (sc-40; Santa Cruz Biotechnology), anti-AIM-1 (BD611082; BD Biosciences), anti-Vinculin (sc-73614; Santa Cruz Biotechnology), anti-AurB-pT232 (636 102; BioLegend), anti-PLK1 (ab17057; Abcam), anti-mCherry (ab125096; Abcam), anti-Bub3 (611 730; BD Biosciences), ani-CENP-E (A15263; ABclonal), Cyclin B (sc-245; Santa Cruz Biotechnology), anti-pH3 (06–570; EMD Millipore), anti-pan-Phospho-serine (AP0932; ABclonal), and anti-Cdc25C (A12234, ABclonal).

Techniques: Immunostaining, Expressing, Blocking Assay, Transfection, Luciferase

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: AurkB-AcK250–BubR1 pathway coordinates the unattachment signal to the maintenance of fibrous corona and SAC. (A, B) Effects of AurkB-mediated phosphorylation and subsequent K250 acetylation in fibrous corona expansion. ( A ) SIM images of anti-Bub1 or anti-MAD2 immunostaining in nocodazole-treated cells are shown. The indicated BubR1 constructs were transfected with siBubR1 targeting the 3′ UTR 48 h before fixation, and nocodazole (200 ng/ml) was applied for 20 h. Volumes were measured from the immunofluorescence assays shown. Number of cells scored: Bub1 in WT, n = 63; Bub1 in S16A, n = 64; Bub1 in S39A, n = 70; MAD2 in WT, n = 196; MAD2 in S16A, n = 196; MAD2 in S39A, n = 69 (mean ± s.e.m.). ( B ) SIM images of anti-ZW10 immunostaining in nocodazole-treated cells. Endogenous BubR1 was depleted via co-transfection with siBubR1. Nocodazole (200 ng/ml) was administered for 20 h, two days post transfection. Insets show enlarged images of ZW10 and CREST staining. Scale bar, 5 μm. ( C ) Volume of ZW10 measured from the immunofluorescence assay shown in panel (B). Number of cells scored: WT, n = 27; S16A, n = 29; S39A, n = 27; S16A;K250Q, n = 31; S39A;K250Q, n = 32; K250Q, n = 29; S16D, n = 31; S39D, n = 32; S16D;K250R, n = 27; S39D;K250R, n = 27; K250R, n = 27 (mean ± s.e.m.). ( D ) Model illustrating the coordination of AurkB-mediated phosphorylation and K250 acetylation in spindle checkpoint signaling. At unattached kinetochores, AurkB phosphorylates BubR1 at S39 (and S16 as well), promoting BubR1 acetylation at K250 by PCAF. This acetylation ensures the maintenance of the RZZ complex and MCC. It also sustains CENP-E at the kinetochore, preparing lateral to end-on attachment (left). Upon successful end-on capture, PP2A activity recruited by AcK250–BubR1 antagonizes AurkB activity, stabilizing KT-MT attachment. Simultaneously, BubR1 immediately deacetylates and degrades through APC/C-mediated ubiquitination, resulting in MCC disassembly and SAC silencing.

Article Snippet: Commercially sourced antibodies used in this study include anti-BubR1 (612 503; BD Biosciences), anti-α-tubulin-FITC (F2168; Upstate), anti-Centromere protein antibody (15–235; Antibodies Inc.), anti-HDAC2 (ab12169; Abcam), anti-HDAC3 (ab7030; Abcam), anti-CDC20 (A15656; ABclonal), anti-MAD2 (A11469; ABclonal), anti-Cyclin B (sc-245; Santa Cruz Biotechnology), anti-β-actin (A700-057; Bethyl), anti-CDC27 (BD 610455; BD Biosciences), anti-FLAG (F1804; Sigma–Aldrich), anti-GAPDH (#2118; Cell Signaling Technology), anti-CENP-A (ab13939;Abcam), anti-ZW10 (sc-81430; Santa Cruz Biotechnology), anti-Myc (sc-40; Santa Cruz Biotechnology), anti-AIM-1 (BD611082; BD Biosciences), anti-Vinculin (sc-73614; Santa Cruz Biotechnology), anti-AurB-pT232 (636 102; BioLegend), anti-PLK1 (ab17057; Abcam), anti-mCherry (ab125096; Abcam), anti-Bub3 (611 730; BD Biosciences), ani-CENP-E (A15263; ABclonal), Cyclin B (sc-245; Santa Cruz Biotechnology), anti-pH3 (06–570; EMD Millipore), anti-pan-Phospho-serine (AP0932; ABclonal), and anti-Cdc25C (A12234, ABclonal).

Techniques: Phospho-proteomics, Immunostaining, Construct, Transfection, Immunofluorescence, Cotransfection, Staining, Activity Assay, Ubiquitin Proteomics

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days after doxycycline (Dox) addition (mean and SD, two-tailed Student’s t test, CENP-C WT cells: n = 6; CENP-C ΔM12BD cells: n = 6; ** p < 0.01). (B) Representative images of DAPI-stained K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Scale bar, 50 μm. (C) Population of normal interphase cells, mitotic cells, and cells with micronuclei in K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Error bars indicate SEM. n = 3 independent experiments; 200 cells from each cell line were quantified in each experiment. (D) The growth curve of K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. K562 WT or CENP-C ΔM12BD cells were treated with or without Dox ( KIF18A OFF or ON). The cell numbers were normalized to those at time 0 of each line. (E) Cell-cycle distribution of conditional knockout of KIF18A in K562 WT cells at each day after Dox addition, based on FACS analysis. (F) Cell-cycle distribution of conditional knockout of KIF18A in K562 CENP-C ΔM12BD cells at each day after Dox addition, based on FACS analysis. (G and H) Quantification of cells with misaligned chromosomes in K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. The experimental scheme is shown. Cells were stained with antibodies against MAD2 (red) to detect misaligned chromosomes and CENP-T (green) as a kinetochore marker. DNA was stained with DAPI (blue). Arrowheads show typical MAD2-positive unaligned chromosomes. Scale bar, 10 μm. The cells with MAD2-positive chromosomes were quantified (H) (mean and SEM, two-tailed Student’s t test; n = 5 independent experiments; n.s., non-significant; ** p < 0.01). (I) Numbers of MAD2 positive kinetochores in each cell in each condition (WT KIF18A ON; WT KIF18A OFF; CENP-C ΔM12BD KIF18A ON; CENP-C ΔM12BD KIF18A OFF) (Mean and SEM, n = 5 independent experiments).

Article Snippet: Mouse anti-human MAD2 , Santa Cruz Biotechnology , Cat#sc-65492; RRID: AB_831526.

Techniques: Knock-Out, Two Tailed Test, Staining, Marker