Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Time‐dependent changes in systolic blood pressure ( SBP ) following angiotensin II (Ang II ) infusion in mice. A , Time course of SBP levels in Ang II –stimulated BubR1 low‐expression ( BubR1 L/L ) mice and wild‐type ( BubR1 +/+ ) mice from days 0 to 6 of Ang II infusion. n=10 BubR1 +/+ mice; n=11 BubR1 L/L mice. B , SBP levels at day 1 and day 6 of Ang II infusion. * P <0.01 vs BubR1 +/+ mice.

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies, including monoclonal antibodies against BubR1 (Novus Biologicals, Littleton, CO; NBP1‐19555), Agtr1 (Abcam, Cambridge, UK; ab18801), Nox4 (Abcam; ab60940), JNK (Jun N‐terminal kinase; Abcam; ab9252), α‐tubulin (Abcam; ab4074), and Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Histological analysis of tissues from BubR1 low‐expression ( BubR1 L/L ) mice and wild‐type ( BubR1 +/+ ) mice after 1 week of angiotensin II (Ang II ) infusion. A , Representative Sirius red‐stained kidney sections. Magnification, ×40; n=10 BubR1 +/+ mice; n=8 BubR1 L/L mice; scale bar, 100 μm. B , Perivascular fibrotic lesion area in the kidney, quantified using morphometry. * P <0.01 vs BubR1 +/+ mice. C , Representative Elastica van Gieson–stained sections of the thoracic aorta. Magnification, ×40; n=12 BubR1 +/+ mice; n=11 BubR1 L/L mice; scale bar, 100 μm. D , Area of aorta media, quantified using morphometry. * P <0.01 vs BubR1 +/+ mice. E , Representative Masson trichrome–stained sections of the heart. Magnification, ×40; n=12 BubR1 +/+ mice; n=11 BubR1 L/L mice; scale bar, 100 μm. F , Ejection fraction, evaluated by echocardiographs; n=12 control mice; n=11 BubR1 L/L mice. * P <0.01 vs BubR1 +/+ mice.

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies, including monoclonal antibodies against BubR1 (Novus Biologicals, Littleton, CO; NBP1‐19555), Agtr1 (Abcam, Cambridge, UK; ab18801), Nox4 (Abcam; ab60940), JNK (Jun N‐terminal kinase; Abcam; ab9252), α‐tubulin (Abcam; ab4074), and Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Expressing, Staining, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Immunohistochemistry of kidneys from BubR1 +/+ and BubR1 L/L mice before and after 1 week of angiotensin II (Ang II ) infusion. A through C , Images of representative kidney sections that were immunostained for BubR1 ( A ), Ang II receptor type 1 ( AGTR 1) ( B ), or Ang II receptor type 2 ( AGTR 2) ( C ). Magnification, ×40; scale bar, 50 μm. BubR1, AGTR 1, and AGTR 2 expression levels in kidney with or without BubR1 reduction, with or without angiotensin II (Ang II ) stimulation (n=5/group). * P <0.01. kpxls indicates kilo pixels; n.s., not significant.

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies, including monoclonal antibodies against BubR1 (Novus Biologicals, Littleton, CO; NBP1‐19555), Agtr1 (Abcam, Cambridge, UK; ab18801), Nox4 (Abcam; ab60940), JNK (Jun N‐terminal kinase; Abcam; ab9252), α‐tubulin (Abcam; ab4074), and Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Immunohistochemistry, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: In vitro assay of BubR1 reduction in human renal proximal tubule cells ( RPTC s). A , BubR1 levels in RPTC s that were treated with siBubR1 or scrambled control si RNA (3 biological replicates each). *P <0.01. B , Angiotensin II (Ang II ) receptor type 1 ( AGTR 1) expression levels in RPTC s with or without BubR1 reduction, with or without 10 μmol/L Ang II stimulation for 24 hours (n=3/group). * P <0.01. siBubR1 indicates short interfering RNA targeting BubR1 .

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies, including monoclonal antibodies against BubR1 (Novus Biologicals, Littleton, CO; NBP1‐19555), Agtr1 (Abcam, Cambridge, UK; ab18801), Nox4 (Abcam; ab60940), JNK (Jun N‐terminal kinase; Abcam; ab9252), α‐tubulin (Abcam; ab4074), and Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: In Vitro, Control, Expressing, Small Interfering RNA

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Expression levels of Nox 4, JNK , and phospho‐ JNK in human renal proximal tubule cells ( RPTC s), with or without angiotensin II (Ang II ) stimulation. A , Representative Western blot of Nox 4, JNK , and phospho‐ JNK / JNK in siBubR1‐ or scramble si RNA control–treated RPTC s, with or without Ang II stimulation. B through D , Quantified expression levels of Nox 4 ( B ), JNK ( C ), and phospho‐ JNK / JNK ( D ) from the blot in ( A ) (n=6/group). * P <0.01. JNK indicates Jun N‐terminal kinase; Nox4, nicotinamide adenine dinucleotide phosphate oxidase‐4; n.s., not significant; p‐JNK, phospho‐JNK; siBubR1, short interfering RNA targeting BubR1 .

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies, including monoclonal antibodies against BubR1 (Novus Biologicals, Littleton, CO; NBP1‐19555), Agtr1 (Abcam, Cambridge, UK; ab18801), Nox4 (Abcam; ab60940), JNK (Jun N‐terminal kinase; Abcam; ab9252), α‐tubulin (Abcam; ab4074), and Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Expressing, Western Blot, Control, Small Interfering RNA

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Attenuation of Angiotensin II –Induced Hypertension in BubR1 Low‐Expression Mice Via Repression of Angiotensin II Receptor 1 Overexpression

doi: 10.1161/JAHA.118.011911

Figure Lengend Snippet: Immunohistochemistry of kidneys from BubR1 +/+ and BubR1 L/L mice before and after 1 week of angiotensin II (Ang II ) infusion. Representative immunohistochemistry sections of Nox4 ( A ) and JNK ( B ) expression. Scale bar, 100 μm. Nox4 and SAPK / JNK expression levels in kidney with or without BubR1 reduction, with or without Ang II stimulation (n=5/group). Quantified expression levels of Nox 4 ( C ), and SAPK / JNK ( D ). * P <0.01. JNK indicates Jun N‐terminal kinase; Nox4, nicotinamide adenine dinucleotide phosphate oxidase‐4; n.s., not significant; SAPK, stress‐activated protein kinase.

Article Snippet: Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies, including monoclonal antibodies against BubR1 (Novus Biologicals, Littleton, CO; NBP1‐19555), Agtr1 (Abcam, Cambridge, UK; ab18801), Nox4 (Abcam; ab60940), JNK (Jun N‐terminal kinase; Abcam; ab9252), α‐tubulin (Abcam; ab4074), and Agtr2 (Santa Cruz Biotechnology; sc‐9040).

Techniques: Immunohistochemistry, Expressing

Journal: Nature communications

Article Title: The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

doi: 10.1038/ncomms6563

Figure Lengend Snippet: Figure 1 | Recruitment of Cdc20 to kinetochores requires BubR1. (a) Schematic of protocol used to synchronize cells and deplete specific proteins using RNAi. (b) Cells depleted of the indicated proteins were stained for CRESTand Cdc20 and BubR1 or Mad2 to check for depletion efficiency. Scale bar, 5 mm. (c) Quantification of Cdc20, BubR1 and Mad2 kinetochore levels in cells treated with the indicated RNAi oligos. The fluorescence from the three z-stacks (200 nm apart) encompassing the bulk kinetochore fluorescent intensity was used and normalized to the CREST signal. Cells were co-stained for Mad2 or BubR1 to ensure that only cells with efficient depletion were analysed. At least 80 kinetochore pairs from eight cells were quantified and the mean and s.e.m. is indicated. (d) Stable HeLa cell line expressing Venus-Cdc20 was treated with the indicated RNAi oligos and the localization of Venus-Cdc20 followed by time-lapse microscopy. Scale bar, 10 mm. (e) Quantification of Venus-Cdc20 at kinetochores in the movies in (d). The signal was quantified from a single z-section and in the frame right after NEBD. A total number of 50 kinetochores from 10 cells were analyzed for each condition and the mean and s.e.m. indicated. An unpaired t-test was performed for statistical analysis (****Po0.0001) and compared with the control-treated cells.

Article Snippet: The following antibodies were used for western blot, immunofluorescence or immunoprecipitation as indicated: a-tubulin 11H10 (rabbit, Cell Signaling) 1:100 IF, b-actin AC-15 (mouse, Abcam) 1:5,000 WB, APC1 A301– 653A (rabbit, Bethyl) 1:500 WB, APC3 35/CDC27 (mouse, BD Biosciences) 1:250 WB, APC4 (mouse, raised against a C-terminal peptide) 5 mg IP, APC7 A302–551A (rabbit, Bethyl) 1:1,000 WB, Bub3 clone 31 (mouse, BD Transduction Laboratories) 1:500 WB, BubR1 A300–995A (rabbit, Bethyl) 1:500 WB, BubR1 A300–386A (rabbit, Bethyl) 1:200 IF, Cdc20 AR12 (mouse, Millipore) 1:200 IF/5 mg IP, Cdc20 E-7 (mouse, Santa Cruz Biotechnology) 1:500 WB, Cdc20 A301–180A (rabbit, Bethyl) 1:1,000 WB, CREST (human, Antibodies Inc.) 1:400 IF, FLAG M2 (mouse, Sigma) 1:5,000 WB, GFP clones 7.1 and 13.1 (mouse, Roche) 1:5,000 WB /1:200 IF, Mad2 from G. Kops (rabbit) 1:200 IF, Mad2 A300–301A (rabbit, Bethyl) 1:1,000 WB, p31 rabbit antibody was generated using full-length p31 as antigen and affinity purified.

Techniques: Staining, Expressing, Time-lapse Microscopy, Control

Journal: Nature communications

Article Title: The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

doi: 10.1038/ncomms6563

Figure Lengend Snippet: Figure 3 | The internal Cdc20 binding site of BubR1 recruits Cdc20 to kinetochores. (a) Schematic of human BubR1 and the location of Cdc20 binding sites and Bub3 binding site as well as the pseudo-kinase domain. Alignment of the region encompassing residues 530–535 of human BubR1 is shown on top and the different constructs used are indicated below. The BubR1 KEN/AAA has the first KEN-box mutated to AAA. (b) Stable HeLa cell lines expressing the different Venus-BubR1 siRNA resistant constructs were used to determine the domains in BubR1 required for Cdc20 kinetochore localization. In brief, cells were treated with a control RNAi oligo (Luciferase) or a BubR1 RNAi oligo and then arrested in mitosis using nocodazole treatment and the proteasome inhibitor, MG132. BubR1 RNAi-treated cells were complemented with the indicated Venus-BubR1 constructs. Cells were stained for BubR1, CREST and Cdc20. Scale bar, 5 mm (c,d). The kinetochore levels of BubR1 (c) and Cdc20 (d) normalized to CREST in control and BubR1-depleted cells. At least 80 kinetochore pairs from eight different cells were analysed, and the mean and s.e.m. are indicated. (e) The level of Cdc20 at kinetochores in cells complemented with the indicated Venus-BubR1 constructs was determined. Only cells with endogenous levels of BubR1 at kinetochores were used for this analysis. At least 80 kinetochore pairs from eight different cells were analysed, and the mean and s.e.m. are indicated. (f) Binding of Cdc20 and Cdc20 4A to BubR1 516–715 was determined by binding 10 mg recombinant FLAG-HA-BubR1 516–715 to FLAG affinity beads and incubating these with increasing concentrations of Strep-His tagged Cdc20 or Cdc20 4A expressed and purified from HEK293 cells. The beads were washed and bound proteins were eluted and analysed by western blot. (g) Quantification of western blot in (f) using Licor technology. Experiment in (f,g) is representative of two independent experiments.

Article Snippet: The following antibodies were used for western blot, immunofluorescence or immunoprecipitation as indicated: a-tubulin 11H10 (rabbit, Cell Signaling) 1:100 IF, b-actin AC-15 (mouse, Abcam) 1:5,000 WB, APC1 A301– 653A (rabbit, Bethyl) 1:500 WB, APC3 35/CDC27 (mouse, BD Biosciences) 1:250 WB, APC4 (mouse, raised against a C-terminal peptide) 5 mg IP, APC7 A302–551A (rabbit, Bethyl) 1:1,000 WB, Bub3 clone 31 (mouse, BD Transduction Laboratories) 1:500 WB, BubR1 A300–995A (rabbit, Bethyl) 1:500 WB, BubR1 A300–386A (rabbit, Bethyl) 1:200 IF, Cdc20 AR12 (mouse, Millipore) 1:200 IF/5 mg IP, Cdc20 E-7 (mouse, Santa Cruz Biotechnology) 1:500 WB, Cdc20 A301–180A (rabbit, Bethyl) 1:1,000 WB, CREST (human, Antibodies Inc.) 1:400 IF, FLAG M2 (mouse, Sigma) 1:5,000 WB, GFP clones 7.1 and 13.1 (mouse, Roche) 1:5,000 WB /1:200 IF, Mad2 from G. Kops (rabbit) 1:200 IF, Mad2 A300–301A (rabbit, Bethyl) 1:1,000 WB, p31 rabbit antibody was generated using full-length p31 as antigen and affinity purified.

Techniques: Binding Assay, Construct, Expressing, Control, Luciferase, Staining, Recombinant, Western Blot

Journal: Nature communications

Article Title: The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.

doi: 10.1038/ncomms6563

Figure Lengend Snippet: Figure 5 | The IC20BD is required for SAC silencing. (a) Stable HeLa cells were depleted of endogenous BubR1 or treated with control RNAi oligo (Luciferase). BubR1 knockdown was complemented by expression of Venus-BubR1, Venus-BubR1 DBub3 or Venus-BubR1 DBub3/D490–560 as indicated and mitotic progression was followed by time-lapse microscopy. Each circle represents a single cell analysed (at least 40 cells were analysed for each condition) and the median (m) is shown as a red bar. Statistical analysis was performed using a Mann–Whitney test. (b) Stable HeLa cell lines were depleted of endogenous BubR1 and complemented by expression of Venus-BubR1 or Venus-BubR1 D490–560 as indicated. Cells were arrested in mitosis with 100 nM nocodazole before filming and subsequently treated with 0.5 mM reversine to silence the SAC. Each circle represents a single cell analysed and at least 120 cells were analysed per condition. Red bars indicate means (m) and a t-test was used for statistical analysis. (c) Representative still images from (b). Scale bar, 10 mm. (d,e) Venus-Cdc20 was purified from a stable HeLa cell line arrested in mitosis with nocodazole. The beads were incubated with increasing concentrations of recombinant FLAG-HA-BubR1 516–715 at room temperature for 1 h and afterwards washed. The binding of endogenous BubR1 and Mad2 to Venus-Cdc20 was analysed by western blot analysis and quantified using Licor technology. Representative of two independent experiments. (f) HeLa cells were transfected with the indicated BubR1 constructs and before filming 200 nM taxol was added. The time from NEBD to mitotic exit was measured by analysing the time-lapse movies and at least 50 cells were analysed per condition. Medians (m) are shown as red bars and a Mann–Whitney test was used for statistical analysis.

Article Snippet: The following antibodies were used for western blot, immunofluorescence or immunoprecipitation as indicated: a-tubulin 11H10 (rabbit, Cell Signaling) 1:100 IF, b-actin AC-15 (mouse, Abcam) 1:5,000 WB, APC1 A301– 653A (rabbit, Bethyl) 1:500 WB, APC3 35/CDC27 (mouse, BD Biosciences) 1:250 WB, APC4 (mouse, raised against a C-terminal peptide) 5 mg IP, APC7 A302–551A (rabbit, Bethyl) 1:1,000 WB, Bub3 clone 31 (mouse, BD Transduction Laboratories) 1:500 WB, BubR1 A300–995A (rabbit, Bethyl) 1:500 WB, BubR1 A300–386A (rabbit, Bethyl) 1:200 IF, Cdc20 AR12 (mouse, Millipore) 1:200 IF/5 mg IP, Cdc20 E-7 (mouse, Santa Cruz Biotechnology) 1:500 WB, Cdc20 A301–180A (rabbit, Bethyl) 1:1,000 WB, CREST (human, Antibodies Inc.) 1:400 IF, FLAG M2 (mouse, Sigma) 1:5,000 WB, GFP clones 7.1 and 13.1 (mouse, Roche) 1:5,000 WB /1:200 IF, Mad2 from G. Kops (rabbit) 1:200 IF, Mad2 A300–301A (rabbit, Bethyl) 1:1,000 WB, p31 rabbit antibody was generated using full-length p31 as antigen and affinity purified.

Techniques: Control, Luciferase, Knockdown, Expressing, Time-lapse Microscopy, MANN-WHITNEY, Incubation, Recombinant, Binding Assay, Western Blot, Transfection, Construct

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation.

doi: 10.1038/modpathol.2012.242

Figure Lengend Snippet: Figure 7 Pathway analysis. Gene Set Enrichment Analysis (GSEA) of malignant peripheral nerve sheath tumors (MPNSTs) and neurofibromas (NFs) from GSE14038 demonstrates enrichment for higher expression of genes related the G2-M checkpoint and chromosomal condensation in malignant peripheral nerve sheath tumors. For example, genes in the expression neighborhood of BUB1B and CENPF are highly upregulated in malignant peripheral nerve sheath tumors. Additionally, many genes in the G2-M checkpoint and the reactome mitotic prometaphase are upregulated in malignant peripheral nerve sheath tumors.

Article Snippet: Commercially available antibodies against PBK, BUB1B and NEK2 were used (PBK: Cell Signaling, polyclonal rabbit antibody, cat. # 4942, 1:50; BUB1B: BD Transduction Laboratories, monoclonal mouse antibody, cat. # 612503, 1:50; NEK2: Abcam, monoclonal mouse antibody, cat. # ab55550, 1:75).

Techniques: Expressing

Journal: Nature Communications

Article Title: Lack of Diaph3 relaxes the spindle checkpoint causing the loss of neural progenitors

doi: 10.1038/ncomms13509

Figure Lengend Snippet: (a – d ) Immunostaining for BubR1 (green) and pHH3 (red) of cortical sections from WT ( a ) and Diaph3 ko ( c ) E10.5 embryos. ( b , d ) Enlargements of the boxed areas in a , c , respectively. BubR1 (a hallmark of SAC activation) accumulated more in normal than in Diaph3-deficient cells (quantification in e ; n =1,473 control cells from 3 animals and 1,144 ko cells from 3 animals; P <0.0001, z -test). ( f ) The density of mitotic cells in the cortex of E10.5 embryos was lower in the ko than in WT ( n =48 WT and 49 ko of 100 μm wide cortical stripes from 3 animals each genotype; P <0.0001; Student’s t -test). ( g ) Quantification of postmetaphasic cells in the population of mitotic cells. The percentage of mitotic cells that underwent the metaphase–anaphase transition was higher in the ko than in control littermates ( n =786 control cells from 6 animals and 889 ko cells from 5 animals; P <0.0001, z -test). ( h , i ) Reduction of BubR1 protein levels in the mutant telencephalon detected by western blotting and quantified relatively to tubulin. The same membranes were blotted for Diaph3 to confirm the absence of the protein ( h ). The level of BubR1 decreased by half in the ko ( i ; n =15 embryos in 4 pools for each genotype). ( j ) Western blotting (WB) detects BubR1 on IP with anti-Diaph3 antibodies from TG cortical lysates. Diaph3 was used as a positive control for the IP (Input). No signal was found in the eluted fraction (flow-through, FT) in presence of Diaph3 antibodies. Detection of the protein in the lysate (Input) required overexposure of the film. Scale bars, 50 μm ( a , c ) and 5 μm ( b , d ). Error bars represent s.e.m.

Article Snippet: The second was obtained following the same strategy, but after insertion of the mouse BubR1 ORF (Origene) upstream of the IRES-EGFP.

Techniques: Immunostaining, Activation Assay, Mutagenesis, Western Blot, Positive Control

Journal: Nature Communications

Article Title: Lack of Diaph3 relaxes the spindle checkpoint causing the loss of neural progenitors

doi: 10.1038/ncomms13509

Figure Lengend Snippet: ( a ) Molecular interactions between Diaph3, CPC and SAC proteins during cell division. Documented interactions are depicted in red and new findings are shown in purple. Diaph3 localizes with CPC proteins and co-immunoprecipitates with Survivin and BubR1. ( b ) Absence of Diaph3 probably disrupts the Diaph3–APC–Eb1 complex, which interacts with and stabilizes the CPC complex at the spindle–kinetochore interface. Loss of Diaph3 impairs the localization of CPC proteins and the ability of dividing cells to activate the SAC. Diaph3-deficient cells fail to accumulate BubR1, leading to slippage to anaphase and mitotic catastrophe and/or cell death of aneuploid progeny.

Article Snippet: The second was obtained following the same strategy, but after insertion of the mouse BubR1 ORF (Origene) upstream of the IRES-EGFP.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: BUBR1 and Closed MAD2 (C-MAD2) Interact Directly to Assemble a Functional Mitotic Checkpoint Complex

doi: 10.1074/jbc.M111.238543

Figure Lengend Snippet: C-MAD2 prolongs the lag in APC/C activation in mitotic cell extracts. A, mitotic extracts prepared from checkpoint-active HeLa cells and incubated in the absence (control) or presence of untagged recombinant MAD2L13A. MAD2L13A was added at 2× and 20× above the concentration of endogenous MAD2 in the extract (∼23 nm). APC/C activity was monitored by degradation of endogenous cyclin B1 and securin. Samples were taken at the indicated minutes after incubation and analyzed by immunoblotting. ATM and an APC/C subunit CDC27 were loading controls. B, quantitation of cyclin B1 degradation shown in A. Cyclin B1 levels were quantified and normalized against ATM levels in scanned Western blots, and the mean ± S.D. (error bars) from two experiments were plotted versus time. C, mitotic extracts incubated in the absence (control) or presence of 20× recombinant MAD2L13A and samples at different times and immunoprecipitated (IP) with anti-CDC27 antibody and probed for MCC and APC/C subunits. D, quantitation of experiment shown in C. BUBR1 and MAD2 levels were normalized against CDC16 (another APC/C subunit) in CDC27 immunoprecipitates and were plotted versus time. The level at the 0 time point in the control experiment was set at 100%. E, kinetics of cyclin B1 degradation in mitotic extracts prepared from untransfected cells (control) or cells transfected with GST-MAD2L13A. GST-MAD2 and endogenous MAD2 were detected simultaneously for comparison of levels. F, untagged recombinant MAD2WT and MAD2ΔC10 added to the mitotic extracts and the rates of cyclin B1 degradation compared.

Article Snippet: Human full-length BUBR1, MAD2, and CDC20 cDNAs were amplified from a prostate cDNA library (Invitrogen) or freshly prepared reverse transcribed cDNAs provided by Dr. Douglas Leaman (University of Toledo).

Techniques: Activation Assay, Incubation, Recombinant, Concentration Assay, Activity Assay, Western Blot, Quantitation Assay, Immunoprecipitation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: BUBR1 and Closed MAD2 (C-MAD2) Interact Directly to Assemble a Functional Mitotic Checkpoint Complex

doi: 10.1074/jbc.M111.238543

Figure Lengend Snippet: The R133E/Q134A mutant reduces C-MAD2 binding to BUBR1 but not CDC20. A, purified recombinant proteins were incubated at intracellular concentrations of endogenous proteins (Fig. 3 legend) for 1 h. GST pulldowns and inputs were probed with the indicated antibodies. Both GST-BUBR1(1–371) and GST-CDC20(111–138) were detected with anti-GST antibody. B, comparison of the full-length BUBR1 binding to MAD2L13A and MAD2LARQ is shown. C, human BUBR1 structure and two models of MCC architecture are illustrated schematically. The two CDC20 binding domains of BUBR1 are shown as CDC20 BD1 and CDC20 BD2. One or both KEN boxes in the N-terminal region of BUBR1 (1–371 residues) are required for CDC20 binding. The GLEBS motif is required for BUBR1-BUB3 interaction. The αC helix of C-MAD2 is represented by a zigzag line.

Article Snippet: Human full-length BUBR1, MAD2, and CDC20 cDNAs were amplified from a prostate cDNA library (Invitrogen) or freshly prepared reverse transcribed cDNAs provided by Dr. Douglas Leaman (University of Toledo).

Techniques: Mutagenesis, Binding Assay, Purification, Recombinant, Incubation

Journal: The Journal of Biological Chemistry

Article Title: BUBR1 and Closed MAD2 (C-MAD2) Interact Directly to Assemble a Functional Mitotic Checkpoint Complex

doi: 10.1074/jbc.M111.238543

Figure Lengend Snippet: BUBR1 directly binds to C-MAD2. A, purified recombinant proteins were incubated at the indicated -fold over previously reported intracellular concentrations ([BUBR1] = 127 nm; [CDC20] = 285 nm; [MAD2] = 230 nm]) (24). GST pulldowns were washed and probed alongside inputs with the indicated antibodies. B, comparison of full-length BUBR1 and BUBR1(1–371) in binding to MAD2L13A is shown. C, HeLa cells co-transfected with GST-MAD2W75A and either GFP or GFP-BUBR1(1–371) were arrested in mitosis with nocodazole. The lysates and GST pulldowns were probed for CDC20, MAD1, GST-MAD2, GFP, and GFP-BUBR1(1–371). D, yeast two-hybrid assay was performed. The yeast strain SKY48 harboring different combinations of baits and preys were tested for growth on galactose-containing leucine-dropout plates. The arrowheads indicate the combinations that produced colonies. E, recombinant MAD2WT was preincubated with GST-CDC20(111–138) immobilized on GSH-agarose beads (+) or beads alone (−) for 24 h. After incubation, GSH-agarose beads were removed by centrifugation, and the resulting supernatants were transferred to new binding reactions containing GST-BUBR1(1–371). GST pulldowns and inputs for the new reactions were probed with the indicated antibodies.

Article Snippet: Human full-length BUBR1, MAD2, and CDC20 cDNAs were amplified from a prostate cDNA library (Invitrogen) or freshly prepared reverse transcribed cDNAs provided by Dr. Douglas Leaman (University of Toledo).

Techniques: Purification, Recombinant, Incubation, Binding Assay, Transfection, Y2H Assay, Produced, Centrifugation