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; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or 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; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or 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; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or 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; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or 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; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or 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; monoclonal antibodies against BubR1 (Novus Biologicals; NBP1‐19555), Agtr1 (Abcam; ab18801), Nox4 (Abcam; ab60940), JNK (Abcam; ab9252), α‐tubulin (Abcam; ab4074), or 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: bioRxiv

Article Title: A mechanism that integrates microtubule motors of opposite polarity at the kinetochore corona

doi: 10.1101/2023.04.25.538277

Figure Lengend Snippet: ( A ) Organization of the human kinetochore and corona. ( B ) Drawing depicting the hierarchical organization of outer kinetochore and kinetochore corona components. Thick arrows indicate recruitment of a protein to the protein indicated by the arrowhead. Thin arrows indicate phosphorylation. The white arrow indicates polymerization. ( C ) Representative images of the localization of CENP-E after depletion of Zwilch and BUBR1. Zwilch RNAi treatment was performed with 100 nM siRNA for 72 h. 48 h after Zwilch RNAi treatment cells were transfected with 100 nM BUBR1 siRNA. 8 h after transfection, cells were synchronized in G2 phase with 9 μM RO3306 for 15 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM Nocodazole, 10 μM MG132 for an additional hour. CENP-C was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 5 μm. ( D ) Quantification of CENP-E levels at kinetochores of the experiment shown in panel C. n refers to individually measured kinetochores. ( E ) Organization of CENP-E with coiled-coil prediction. ( F ) Representative images showing the localization of different EGFP CENP-E constructs in prometaphase after depletion of CENP-E with 60 nM siRNA. 13 h after RNAi treatment cells were electroporated with electroporation buffer or recombinant EGFP CENP-E constructs as indicated. Following an 8 h recovery, cells were synchronized in G2 phase with 9 μM RO3306 for 15 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM Nocodazole for an additional hour. CENP-C was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 5 μm. ( G ) Quantification of EGFP levels at kinetochores of the experiment shown in panel F. n refers to individually measured kinetochores. ( H ) mCH RZZ mCh S ring-binding assays showing the recruitment of various EGFP CENP-E truncations (10 nM concentration) to mCH RZZ mCh S rings (approx. 40 nM concentration). Scale bar: 5 μm. ( I ) RZZS ring-binding assays showing the recruitment of EGFP CENP-E 2070C to mCH RZZ mCh S rings is unaffected by increasing concentrations of BUBR1 KD . Scale bar: 5 μm.

Article Snippet: After blocking with 5% boiled goat serum (BGS) in PHEM buffer for 1 hour, cells were incubated for 2 hours at room temperature with the following primary antibodies: BUB1 (mouse, Abcam, ab54893, 1:400), BUBR1 (rabbit, Thermo Scientific #720297, 1:1000), CENP-C (guinea pig, MBL, #PD030, 1:1000), CENP-E (mouse, Abcam, ab5093, 1:200), CENP-F (rabbit, Novus NB500–101, 1:300), CREST/anti-centromere antibodies (human, Antibodies, Inc., 1:200), Dynactin-p150 glued (mouse, BD Trans.

Techniques: Transfection, Staining, Construct, Electroporation, Recombinant, Binding Assay, Concentration Assay