mad2 protein  (Cell Signaling Technology Inc)

Journal: Molecular Cancer

Article Title: RB acute loss induces centrosome amplification and aneuploidy in murine primary fibroblasts

doi: 10.1186/1476-4598-5-38

Figure Lengend Snippet: Acute loss of Rb leads to chromosome instability and increases MPM2 positive cells . ( A ) pRb deficient MEFs show increased aneuploid metaphases both hypodiploid (<40 chromosomes) and hyperdiploid (>40 chromosomes). Each bar indicates mean + s.e. of three independent experiments. ( B ) pRb deficient MEFs have increased percentage of cells with micronuclei. An average of 100 cells were analyzed for each time point, each bar represents mean + s.e. of two experiments. ( C ) MEFs acutely depleted of pRb (Rb-/-) have increased number of mitotic cells with multiple centrosomes (arrowheads). Mitotic cells and centrosomes were revealed by using primary antibodies against MPM2 (Mitotic Protein Monoclonal) and γ-tubulin, that were detected by FITC (green) and TRITC (red) conjugated secondary antibody respectively. ( D ) Histograms indicating the percentage of MPM2 positive cells in pRb competent and deficient MEFs. An average of 100 cells were analyzed for each time point, each bar represents mean + s.e. of at least two experiments. ( E ) Western blot analysis showing the amount of MAD2 protein after 3 days from the adenoviral infection: Rb LoxP/LoxP MEF uninfected ( lane 1), infected with the empty adenoviral vector ( lane 2) and with the Ad-Cre vector ( lane 3).

Article Snippet: To detect amount of pRb and MAD2 proteins blots were probed with mouse monoclonal antibodies anti pRb (554136, Becton Dickinson) and anti MAD2 (Santa Cruz Biotechnology) respectively as recommended by manufacturer.

Techniques: Western Blot, Infection, Plasmid Preparation

Journal: Molecular Cancer

Article Title: RB acute loss induces centrosome amplification and aneuploidy in murine primary fibroblasts

doi: 10.1186/1476-4598-5-38

Figure Lengend Snippet: Changes in transcript levels of genes associated with mitotic progression after pRb acute loss

Article Snippet: To detect amount of pRb and MAD2 proteins blots were probed with mouse monoclonal antibodies anti pRb (554136, Becton Dickinson) and anti MAD2 (Santa Cruz Biotechnology) respectively as recommended by manufacturer.

Techniques: Binding Assay, Transformation Assay

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: CMLD-2 effects on MAD2 protein levels in thyroid cancer cells. Panel A. MAD2 is identified as HuR target by RIP assay. Thyroid cancer cells were lysed and immunoprecipitated with anti-HuR or anti-IgG antibodies. HuR–bound mRNAs were amplified using specific MAD2 and MARCH3 (negative control) primers and analyzed by qPCR. All samples were run in triplicate. IgG-immunoprecipitate was arbitrarily set at 1.0 and the enrichment was expressed as relative expression value (Fold Enrichment). Panel B. SW1736, 8505 C, BCPAP and K1 cells were treated with DMSO or CMLD-2 35 µM for 72 h. Cells were collected and MAD2 protein levels were analyzed by Western Blot. Panel C. Densitometric analysis of MAD2 protein levels obtained with Western Blot assay. For each cell line, the results were normalized against Actin and expressed as percentage over control. Results are shown as mean ± SD. ***p < 0.001, ****p < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Immunoprecipitation, Amplification, Negative Control, Expressing, Western Blot

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: Biological effects of MAD2 silencing in thyroid cancer cells. 8505 C and BCPAP cells were transfected with vehicle (mock), non-targeting siRNA (NC, negative control) or three different siRNA (#1, #2 and #3) sequence specific to MAD2 (5 nM) for 72 h and cell viability was analyzed by MTT assay. All experiments were run in sixfold. P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Transfection, Negative Control, Sequencing, MTT Assay

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: MAD2 is involved in CMLD-2 effects in thyroid cancer cell lines. Panel A. 8505 C and BCPAP cells were treated with DMSO, CMLD-2, pCMV empty vector (NC, negative control), pCMV vector specific for MAD2 (1.3 µg), alone or in combinations. Cells were collected after 72 h treatment and MAD2 protein levels were analyzed by Western Blot. Panel B. 8505 C and BCPAP cells were treated with DMSO, CMLD-2, pCMV empty vector (NC, negative control), pCMV vector specific for MAD2 (1.3 µg), alone or in combinations and cell viability was determined by MTT assay after 72 h. NC was arbitrarily set at 1 and cell viability was expressed as relative expression value. All samples were run in sixfold. P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Plasmid Preparation, Negative Control, Western Blot, MTT Assay, Expressing

Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Hec1 S69 is phosphorylated throughout mitosis. (A) Amino acid sequences of the human and PtK1 cell Hec1 N-terminal tail domain. Shown in yellow is the human peptide sequence that was used to generate the S69 phosphospecific antibody. The arrow points to S69 in the human sequence and the corresponding serine residue in the PtK1 sequence. Asterisks indicate all other mapped Aurora B kinase sites in the human Hec1 tail domain ( ; ). (B) Immunofluorescence images of HeLa cells stained with phosphospecific antibodies to Hec1 pS69. Depletion of Hec1 (bottom) results in loss of pS69 staining at kinetochores. Cells are also immunostained with antibody 9G3 (pan-Hec1 antibody) and an anticentromere antibody (ACA) derived from human calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum. (C) Immunofluorescence images of HeLa cells demonstrating kinetochore localization of pS69 during mitosis. Quantification is shown on the right. For each phase shown, ≥400 kinetochores from ≥30 cells were measured. (D) Immunofluorescence images of a HeLa cell stained with antibodies to pS69 and Mad2. For the cell shown, most chromosomes are aligned at the spindle equator, and one chromosome remains near a spindle pole (arrows). A schematic illustrating examples of pole-proximal chromosomes is shown on the left. (E) Immunofluorescence images of HeLa cells depleted of CENP-E to increase the number of pole-proximal chromosomes (arrows) and stained with Hec1 phosphospecific antibodies. Quantification is shown on the right from one representative experiment. n values are as follows: pS69, 20 polar kinetochores and 40 aligned kinetochores; pS55, 17 polar kinetochores and 57 aligned kinetochores; and pS44, 13 polar kinetochores and 29 aligned kinetochores. Error bars indicate SD. Bars: (B, C, and E) 10 µm; (D) 3 µm.

Article Snippet: Two rabbits were immunized with the Mad2 protein (Rockland Immunochemicals Inc), and antisera from the two injected rabbits were affinity purified against full-length Mad2 protein using a HiTrap-NHS column.

Techniques: Sequencing, Residue, Immunofluorescence, Staining, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Hec1 S69 is phosphorylated throughout mitosis. (A) Amino acid sequences of the human and PtK1 cell Hec1 N-terminal tail domain. Shown in yellow is the human peptide sequence that was used to generate the S69 phosphospecific antibody. The arrow points to S69 in the human sequence and the corresponding serine residue in the PtK1 sequence. Asterisks indicate all other mapped Aurora B kinase sites in the human Hec1 tail domain ( ; ). (B) Immunofluorescence images of HeLa cells stained with phosphospecific antibodies to Hec1 pS69. Depletion of Hec1 (bottom) results in loss of pS69 staining at kinetochores. Cells are also immunostained with antibody 9G3 (pan-Hec1 antibody) and an anticentromere antibody (ACA) derived from human calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum. (C) Immunofluorescence images of HeLa cells demonstrating kinetochore localization of pS69 during mitosis. Quantification is shown on the right. For each phase shown, ≥400 kinetochores from ≥30 cells were measured. (D) Immunofluorescence images of a HeLa cell stained with antibodies to pS69 and Mad2. For the cell shown, most chromosomes are aligned at the spindle equator, and one chromosome remains near a spindle pole (arrows). A schematic illustrating examples of pole-proximal chromosomes is shown on the left. (E) Immunofluorescence images of HeLa cells depleted of CENP-E to increase the number of pole-proximal chromosomes (arrows) and stained with Hec1 phosphospecific antibodies. Quantification is shown on the right from one representative experiment. n values are as follows: pS69, 20 polar kinetochores and 40 aligned kinetochores; pS55, 17 polar kinetochores and 57 aligned kinetochores; and pS44, 13 polar kinetochores and 29 aligned kinetochores. Error bars indicate SD. Bars: (B, C, and E) 10 µm; (D) 3 µm.

Article Snippet: Two rabbits were immunized with the Mad2 protein (Rockland Immunochemicals Inc), and antisera from the two injected rabbits were affinity purified against full-length Mad2 protein using a HiTrap-NHS column.

Techniques: Sequencing, Residue, Immunofluorescence, Staining, Derivative Assay

Journal: PLoS ONE

Article Title: p31 comet -Induced Cell Death Is Mediated by Binding and Inactivation of Mad2

doi: 10.1371/journal.pone.0141523

Figure Lengend Snippet: (A) A schematic map of human p31 comet deletion mutants of the N- and C-termini, and regions affecting Mad2 binding and nuclear localization signal (NLS). (B) HeLa cells were infected with retroviruses containing full-length p31 comet or the above deletion constructs and evaluated in terms of colony formation. GFP+ signifies colonies expressing p31 comet . The total represents the sum of GFP-positive and -negative colonies. Values are presented as the mean of triplicate samples. (C) Interactions between p31 comet deletion mutants and Mad2 were evaluated using an immunoprecipitation assay. Lysates from cells co-transfected with FLAG-tagged wild-type or deletion constructs of p31 comet and Myc-tagged Mad2 were immunoprecipitated with an anti-FLAG antibody, and the resulting immunoprecipitates probed with an anti-Myc antibody. Expression of recombinant proteins was confirmed using antibodies directed against FLAG or Myc.]

Article Snippet: Mad2 protein in immunoprecipitates was detected using an anti-Myc antibody (2278, Cell Signaling, MA).

Techniques: Binding Assay, Infection, Construct, Expressing, Immunoprecipitation, Transfection, Recombinant

Journal: PLoS ONE

Article Title: p31 comet -Induced Cell Death Is Mediated by Binding and Inactivation of Mad2

doi: 10.1371/journal.pone.0141523

Figure Lengend Snippet: (A) Effects of a p31 comet mutant ( Q83A/F191A ) on apoptosis (lower panel) and repression of colony formation (upper panel) were analyzed in HeLa cells. Expression of p31 comet and Q83A/F191A was confirmed via Western blot. (B) The effects of a p31 comet mutant ( Q83/F191A ) on Myc/Ras -mediated colony formation in p53-/- MEFs were compared with those of wild-type (WT) p31 comet . (C) The effect of Mad2 on p31 comet -induced repression of clonogenic cell survival was analyzed in HeLa cells in the presence of either wild-type or dominant-negative mutant Mad2 ( R133E/Q134A ) (Mad2mt). (D) Expression of p31 comet , Mad2, and R133E/Q134A mutant (Mad2mt) was confirmed using immunoblotting. Values represent mean of triplicate samples. Statistical significance ( p- value) was calculated with Student’s t-test. * indicates p >0.05, when each test transfection was compared with that of empty vector.

Article Snippet: Mad2 protein in immunoprecipitates was detected using an anti-Myc antibody (2278, Cell Signaling, MA).

Techniques: Mutagenesis, Expressing, Western Blot, Dominant Negative Mutation, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: p31 comet -Induced Cell Death Is Mediated by Binding and Inactivation of Mad2

doi: 10.1371/journal.pone.0141523

Figure Lengend Snippet: (A) Nocodazole was added to cultures of HeLa cells transfected with either Mad2 siRNA (si-Mad2) or control siRNA (si-Con) and transduced retrovirally with p31 comet or empty vector. Subsequent morphological changes were compared under a light microscope (x100). (B) Under the above conditions, cell cycle progression was monitored with histograms of the DNA content (2N, 4N and 8N) at 24 h and 36 h after treatment. (C) Apoptosis was analyzed via Annexin V staining in HeLa and Huh7 cells transfected with either Mad2 siRNA (si-Mad2) or control siRNA (si-Con). (D) Mad2 depletion with Mad2 siRNA was confirmed using Western blot.

Article Snippet: Mad2 protein in immunoprecipitates was detected using an anti-Myc antibody (2278, Cell Signaling, MA).

Techniques: Transfection, Control, Plasmid Preparation, Light Microscopy, Staining, Western Blot