Journal: Journal of Cell Science

Article Title: Temporal changes in Hec1 phosphorylation control kinetochore-microtubule attachment stability during mitosis

doi: 10.1242/jcs.072629

Figure Lengend Snippet: Hec1 is phosphorylated at kinetochores on multiple N-terminal serine residues and phosphorylation is dependent on Aurora B kinase. (A) Immunofluorescence images of PtK1 and HeLa cells stained with Hec1 phosphorylation-specific antibodies. Antibodies raised against peptides containing phosphorylated Ser8, Ser44 and Ser55 recognized kinetochores consistently in PtK1 cells, and antibodies raised against peptides containing phosphorylated Ser15, Ser44 and Ser55 recognized kinetochores consistently in HeLa cells. (B) Immunofluorescence images of mock-depleted or Hec1-depleted PtK1 and HeLa cells probed for Ser55-P. Kinetochore localization of the anti-Ser55-P antibody and the Hec1 9G3 antibody is lost upon depletion of Hec1. (C) Immunoblots of recombinantly expressed and purified NDC80Bonsai complexes probed with the non-phosphorylation-specific Hec1 9G3 antibody and the 4 phosphorylation-specific Hec1 antibodies. Purified NDC80Bonsai complexes were incubated with activated Aurora B kinase in the presence or absence of ATP and subjected to SDS-PAGE prior to immunoblot analysis. (D) Immunofluorescence images of a PtK1 and HeLa cell treated with 2 μM ZM447439. Kinetochore localization of Ser44-P is significantly diminished in both cell types in response to treatment with the inhibitor.

Article Snippet: The following commercially available primary antibody dilutions were used: mouse anti-Hec1 9G3 at 1:3000 (Novus Biologicals, Littleton, CO), human anti-centromere antibody at 1:300 (ACA; Antibodies, Inc., Davis, CA), DM1α mouse anti-tubulin at 1:200 (Sigma); rat anti-tubulin at 1:200 (Sigma); and rabbit anti-phosphorylated Aurora-B (Thr232- P ) at 1:1000 (Rockland Immunochemicals, Gilbertsville, PA).

Techniques: Immunofluorescence, Staining, Western Blot, Purification, Incubation, SDS Page

Journal: Journal of Cell Science

Article Title: Temporal changes in Hec1 phosphorylation control kinetochore-microtubule attachment stability during mitosis

doi: 10.1242/jcs.072629

Figure Lengend Snippet: Phosphorylated Aurora B kinase localizes to the centromere and kinetochore during mitosis. (A,B) Immunofluorescence images of PtK1 cells (A) and HeLa cells (B) in various stages of mitosis. Enlarged images and insets in A and B show kinetochore pairs (indicated by the arrows). (C,D) Quantification of phosphorylated Aurora B kinase at the outer kinetochore during mitosis. For each mitotic phase or condition, a minimum of 100 kinetochores were measured from a total of at least 10 cells. (E) Immunofluorescence images of PtK1 cells (top) and HeLa cells (bottom) expressing Aurora-B-kinase–GFP (shown as ‘Aurora B kinase’ and in red for clarity) treated with or without 2 μM ZM447439 prior to fixation and stained with anti-Thr232-P Aurora B kinase antibodies (green). Insets in E show individual representative kinetochore pairs for each condition. Scale bars: 10 μm. Error bars indicate s.d.

Article Snippet: The following commercially available primary antibody dilutions were used: mouse anti-Hec1 9G3 at 1:3000 (Novus Biologicals, Littleton, CO), human anti-centromere antibody at 1:300 (ACA; Antibodies, Inc., Davis, CA), DM1α mouse anti-tubulin at 1:200 (Sigma); rat anti-tubulin at 1:200 (Sigma); and rabbit anti-phosphorylated Aurora-B (Thr232- P ) at 1:1000 (Rockland Immunochemicals, Gilbertsville, PA).

Techniques: Immunofluorescence, Expressing, Staining

Journal: Journal of Cell Science

Article Title: Temporal changes in Hec1 phosphorylation control kinetochore-microtubule attachment stability during mitosis

doi: 10.1242/jcs.072629

Figure Lengend Snippet: Hec1 is not maximally rephosphorylated in response to lack of MT attachment. (A) Immunofluorescence images of control PtK1 and HeLa cells and cells treated with nocodazole prior to fixation. In the top panel of images, an example is given of a PtK1 cell that has a ‘compact’ morphology, indicating that at least partial chromosome alignment occurred prior to incubation with nocodazole, and one that has a ‘dispersed’ morphology, indicating that the cell entered mitosis in the presence of nocodazole. (B) Immunofluorescence images of a PtK1 cell with compact chromosomes treated with nocodazole and stained with an anti-Thr232-P Aurora B kinase antibody. Enlarged images in B show a representative kinetochore pair from this experiment. Scale bar: 10 μm.

Article Snippet: The following commercially available primary antibody dilutions were used: mouse anti-Hec1 9G3 at 1:3000 (Novus Biologicals, Littleton, CO), human anti-centromere antibody at 1:300 (ACA; Antibodies, Inc., Davis, CA), DM1α mouse anti-tubulin at 1:200 (Sigma); rat anti-tubulin at 1:200 (Sigma); and rabbit anti-phosphorylated Aurora-B (Thr232- P ) at 1:1000 (Rockland Immunochemicals, Gilbertsville, PA).

Techniques: Immunofluorescence, Incubation, Staining

Journal: Redox Biology

Article Title: Covalent Aurora A regulation by the metabolic integrator coenzyme A

doi: 10.1016/j.redox.2019.101318

Figure Lengend Snippet: Kinase profiling of CoA, dpCoA and ADP. The ability of CoA, dpCoA or ADP (100 μM final concentration of each compound and the indicated concentrations of ATP) to inhibit the phosphotransferase activity of 117 kinases was determined using a radioactive filter-binding assay at the International Centre for Kinase Profiling, DSTT, University of Dundee. Kinase activity in the absence of inhibitor is taken as 100%.

Article Snippet: The following antibodies were employed: mouse anti-CoA antibody; mouse anti-FLAG M2 antibody (Sigma-Aldrich); rabbit anti-Aurora A and anti-Aurora B antibodies (Merck-Millipore); rabbit anti-pT288 Aurora A (Cell Signaling Technology), Alexa Fluor 680 goat anti-mouse IgG H&L (Life Technologies) and IRdye 800 CW goat anti-rabbit IgG H&L (LI-COR Biosciences).

Techniques: Concentration Assay, Activity Assay, Filter-binding Assay

Journal: Redox Biology

Article Title: Covalent Aurora A regulation by the metabolic integrator coenzyme A

doi: 10.1016/j.redox.2019.101318

Figure Lengend Snippet: Specificity of Aurora A interaction with CoA is controlled by Thr 217. (A) Amino acid conservation in vertebrate Aurora kinases. Thr 217 defines Aurora A, and is changed to a Glu residue in Aurora B and C. Cys 290 (boxed) is invariant in all Aurora kinases, and lies in the activation segment adjacent to the phosphorylated Thr 288 (human Aurora A numbering). (B) In vitro CoAlation of Aurora A. (C) In vitro CoAlation of Aurora A is abolished in the Thr217Glu mutant. (D) In vitro CoAlation is not detected with Aurora B. (E) The Glu161Thr Aurora B mutant is efficiently CoAlated. Experiments were performed with the indicated concentration of CoA. Reaction mixtures were separated by SDS-PAGE and immunoblotted with anti-CoA, Aurora A or Aurora B antibodies. (F) A thermal shift assay was employed to evaluate Aurora A binding to 5 mM ATP, 5 mM CoA or 0.1 mM MLN8237, in the presence and absence of 1 mM DTT, as indicated. Assays were performed with T 288 phosphorylated, active, Aurora A (open bars), kinase-dead, dephosphorylated Aurora A (Asp274Asn, green bars), Cys290Ala Aurora A (red bars) or Thr217Glu Aurora A (blue bars). Mean ΔT m values ± SD (n = 3) were calculated by subtracting the control T m value (buffer, no addition) from the measured T m value. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following antibodies were employed: mouse anti-CoA antibody; mouse anti-FLAG M2 antibody (Sigma-Aldrich); rabbit anti-Aurora A and anti-Aurora B antibodies (Merck-Millipore); rabbit anti-pT288 Aurora A (Cell Signaling Technology), Alexa Fluor 680 goat anti-mouse IgG H&L (Life Technologies) and IRdye 800 CW goat anti-rabbit IgG H&L (LI-COR Biosciences).

Techniques: Activation Assay, In Vitro, Mutagenesis, Concentration Assay, SDS Page, Thermal Shift Assay, Binding Assay

Journal: Redox Biology

Article Title: Covalent Aurora A regulation by the metabolic integrator coenzyme A

doi: 10.1016/j.redox.2019.101318

Figure Lengend Snippet: Oxidative stress induces Aurora A CoAlation in human cells. (A) Aurora A CoAlation is induced in cellular response to H 2 O 2 . FLAG-tagged WT Aurora A and WT Aurora B were transiently overexpressed in HEK293/Pank1β. Transfected cells were treated for 30 min with 0.25 mM H 2 O 2 . Overexpressed proteins were immunoprecipitated with an anti-FLAG antibody and the immune complexes immunoblotted with anti-CoA and anti-FLAG antibodies. (B) Oxidising agents promote Aurora A CoAlation in vivo . FLAG-tagged WT Aurora A was transiently over expressed in HEK293/Pank1β. Transfected cells were treated for 30 min with a panel of oxidising agents (250 μM H 2 O 2 , 500 μM diamide, 50 μM menadione, 10 μM phenylarsine oxide and 1 μM rotenone). Transiently expressed proteins were immunoprecipitated with an anti-FLAG antibody, separated by SDS-PAGE under non-reducing conditions and immunoblotted with anti-CoA antibodies. (C) Phosphorylation at Thr 288 and dimerization of Aurora A are induced in cells by oxidising agents. FLAG-tagged WT Aurora A was transiently overexpressed in HEK293/Pank1β. Transfected cells were treated for 30 min with a panel of oxidising agents (250 μM H 2 O 2 , 500 μM diamide, 50 μM menadione, 10 μM phenylarsine oxide and 1 μM rotenone). Transiently expressed proteins were immunoprecipitated with an anti-FLAG antibody, separated by SDS-PAGE under reducing and non-reducing conditions and immunoblotted with anti-FLAG and anti-pT288 Aurora A antibodies. (D) Schematic illustration showing the key features of the ‘dual anchor’ mechanism for interaction of CoA with Thr 217 and Cys 290 in Aurora A.

Article Snippet: The following antibodies were employed: mouse anti-CoA antibody; mouse anti-FLAG M2 antibody (Sigma-Aldrich); rabbit anti-Aurora A and anti-Aurora B antibodies (Merck-Millipore); rabbit anti-pT288 Aurora A (Cell Signaling Technology), Alexa Fluor 680 goat anti-mouse IgG H&L (Life Technologies) and IRdye 800 CW goat anti-rabbit IgG H&L (LI-COR Biosciences).

Techniques: Transfection, Immunoprecipitation, In Vivo, SDS Page

Journal: The Journal of Cell Biology

Article Title: Notch activates cell cycle reentry and progression in quiescent cardiomyocytes

doi: 10.1083/jcb.200806104

Figure Lengend Snippet: Ad-N2 ICD –infected NRVCs arrest at the G 2 /M interphase. (A and B) 3 d after birth, NRVCs were infected with Ad-N2 ICD or Ad-β-gal, or not infected, and, 12 h later, treated with aphidicolin or nocodazole for an additional 36, 48, or 60 h. For nocodazole+ release, nocodazole was removed during the last 12 h of culture (see schematic in A, top). The percentages of cells in S/G 2 /M phase in MF20 + (cardiomyocytes; A) and MF20 − (noncardiomyocytes; B) populations were determined from flow cytometry. Nocodazole did not increase the incidence of G 2 /M cells among the N2 ICD -treated cardiomyocyte population, which is indicative of a block at the onset of M phase. The example shown is representative of four experiments with similar outcomes. (C–I) NRVCs were either left uninfected (C and F) or infected with Ad-βGal (D and G) or Ad-N2 ICD (E and H), cultured for 48 h, then stained with MF20 (red, Alexa 594) and for AuroraB (green, Alexa 488; C–E) or for phospho-His3 (Ser10; green, Alexa 488; F–H). The percentages of positive cells within the MF20 + population was then determined (I). Nuclear-localized AuroraB but not phospho-His3 was detected in response to N2 ICD . Error bars indicate ±SD. Bar, 25 μm.

Article Snippet: Anti-AuroraB rabbit Ab , 1:250 , BD Biosciences.

Techniques: Infection, Flow Cytometry, Blocking Assay, Cell Culture, Staining

Journal: The Journal of Cell Biology

Article Title: Notch activates cell cycle reentry and progression in quiescent cardiomyocytes

doi: 10.1083/jcb.200806104

Figure Lengend Snippet: Antibodies and dilutions used for immunofluorescent detection of proteins

Article Snippet: Anti-AuroraB rabbit Ab , 1:250 , BD Biosciences.

Techniques: