anti mad2 rabbit polyclonal  (Bethyl)

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Representative images of WT and CAMSAP2 KO spindles. Notably, 2% PFA-fixed cells were stained for α-tubulin (green), Mad2 (red), and DAPI (blue). Scale bar; 10 μm. (B) The spindle length of cells prepared in (A) measured based on α-tubulin signals. Mad2-negative spindles were exclusively chosen for quantification as of metaphase. n = 79 (WT) and 86 (CAMSAP2 KO) cells from four independent experiments were analyzed. Boxplots indicate 25th percentile, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test. (C) Areas of chromosomes in metaphase cells prepared in (A), measured from DAPI signals. Mad2-negative spindles were exclusively chosen for quantification. n = 79 (WT) and 86 (CAMSAP2 KO) cells from four independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test. (D) Orientation of polar microtubules extending inward from the spindle. Representative images acquired with the super-resolution confocal microscope LSM980 Airyscan are shown. Cells were fixed with 2% PFA and stained for α-tubulin (green) and DAPI (blue). The centrosome regions are boxed and enlarged below. Radar charts show distribution of angles of polar microtubules emanating towards chromosomes. Astral microtubules are not included. The origin of microtubules (the spindle pole) was centered in the chart and labels on the radar axis indicate frequencies. The sum of all the frequencies equals 1.0. Each microtubule emanating from the pole (traced in red) is taken to measure its orientation. n = 158 microtubules from 9 cells (WT) and 139 microtubules from 9 cells (CAMSAP2 KO) from four independent experiments. Scale bar; 10 μm. (E) Orientation of astral microtubules as in (D). Boxed regions are enlarged and brightness is adjusted to visualize astral microtubules. Radar charts show distribution of angles of astral microtubules. n = 255 microtubules from 11 cells (WT) and 336 from 12 cells (CAMSAP2 KO) from four independent experiments. Scale bars; 10 μm. (F, G) The length (F) and number (G) of astral microtubules captured in (E) are quantified. For (F), n = 263 microtubules from 9 cells (WT) and 324 from 15 cells (CAMSAP2 KO) from three independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test. For (G), n = 9 cells (WT) and 15 cells (CAMSAP2 KO) from three independent experiments. **P < 0.01, Student’s t-test. (H) Definition of the degree of displaced centrosomes. Cells were fixed with 2% PFA and stained for α-tubulin (green), γ-tubulin (red), and DAPI (blue). The γ-tubulin punctate signals were defined as centrosomes, and the distance between two γ-tubulin dots was defined as the γ-tubulin distance (red bidirectional arrows). The distance between two α-tubulin signals at the tips of the spindle was defined as the α-tubulin distance (green bidirectional arrows). Degree of displaced centrosome was calculated as the ratio of γ-tubulin distance to α-tubulin distance. n = 57 (WT) and 59 (CAMSAP2 KO) cells from three independent experiments. Boxplots indicate 25th, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test. Scale bar; 10 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-CAMSAP3 [ ], a gift from Masatoshi Takeichi (RIKEN, Kobe, Japan), rabbit polyclonal anti-CAMSAP2 (Novus biologicals, Colorado, USA), mouse monoclonal anti-GFP (Roche, Basel, Switzerland), mouse monoclonal anti-γ-tubulin (GTU-88; Sigma-Aldrich), anti-human centromere protein (CREST patient serum; Antibodies Inc, California, USA), anti-KIF2A rabbit polyclonal (Novus biologicals), anti-Mad2 rabbit polyclonal (Bethyl Laboratories, Texas, USA), rabbit monoclonal (for ) and mouse monoclonal ( ) anti-phospho histone H3 (abcam, Cambridge, UK), rabbit monoclonal anti-HAUS6 (abcam), mouse anti-Human BUBR1 (BD Transduction Laboratories™, California, USA), and rabbit polyclonal anti-CAMSAP1 (Novus biologicals).

Techniques: Staining, Microscopy

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Signal intensity of α-tubulin immunostained in WT and CAMSAP2 KO cells quantified along the spindle axis (red lines). Notably, 2% PFA-fixed cells stained for α-tubulin (green) and DAPI (blue). Images analyzed in were used for quantification. Heatmaps indicate gradient fluorescence intensities of α-tubulin signals. Lower graphs show in gray, each spindle; black (WT) and red (CAMSAP2 KO), average of all spindles. Background intensities were subtracted, which were then normalized by the maximum values of the spindles. Mad2-negative spindles were chosen as metaphase ones for quantification. n = 79 (WT) and 86 (CAMSAP2 KO) cells from four independent experiments. Scale bar; 10 μm. (B) Representative images of spindle untreated (control) and cold treated WT and CAMSAP2 KO cells. Notably, 2% PFA-fixed cells were stained for α-tubulin (green), CREST (red), and DAPI (blue). Scale bar; 10 μm. (C) Signal intensities of α-tubulin in spindles of (B). The mean intensity in the spindle excluding poles were measured. The boxed region is shown magnified to the right. The background intensity off the spindle was subtracted. n = 57 (control WT), 71 (cold treated WT), 83 (control CAMSAP2 KO), and 76 (cold treated CAMSAP2 KO) cells from three independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. *P < 0.05, control WT vs cold treated WT: Welch’s t-test, n.s.: P > 0.05, control CAMSAP2 KO vs cold treated CAMSAP2 KO: Student’s t-test. (D) γ-tubulin intensities on the spindle. Notably, 2% PFA-fixed cells were stained for α-tubulin (green), γ-tubulin (red), and DAPI (blue). γ-tubulin signal intensities on the spindle excluding the poles were quantified. The background intensity off the spindle was subtracted. n = 88 (WT) and 76 (CAMSAP2 KO) cells from three independent experiments. Boxplots indicate 25th percentile, median, and 75th percentile values. ****P < 0.0001, Welch’s t-test, Scale bar; 10 μm. (E) Visualization of bridging fibers in the spindle. Representative images of single z-planes acquired with LSM980 Airyscan are shown. Notably, 2% PFA-fixed cells were stained for α-tubulin (green), CREST (red), and DAPI (blue). Insets are enlarged with adjusted brightness, showing sister kinetochore fibers and bridging fibers behind the sister-kinetochore fibers (arrowheads). Frequencies of sister kinetochore pairs accompanying bridging fibers behind. n = 56 pairs of kinetochores from 18 cells (WT) and 53 from 14 cells (CAMSAP2 KO) from four independent experiments. Boxplots indicate 25th, median, and 75th percentile values. *P < 0.05, Student’s t-test. Scale bar; 10 μm. (F) α-tubulin intensity of bridging fibers (left) and kinetochore fibers (right). The intensity was measured according to a previous report with some modifications . For the intensities of bridging fibers (left), 38 fibers from 17 cells (WT) and 15 from 10 cells (CAMSAP2 KO) were obtained from three independent experiments. Pairs without visible bridging fibers were excluded from quantification. For kinetochore fibers (right), 86 kinetochore fibers from 17 cells (WT) and 59 from 13 cells (CAMSAP2 KO) from three independent experiments. Boxplots indicate 25th, median, and 75th percentile values. *P < 0.05, Student’s t-test.

Article Snippet: The following primary antibodies were used: rabbit anti-CAMSAP3 [ ], a gift from Masatoshi Takeichi (RIKEN, Kobe, Japan), rabbit polyclonal anti-CAMSAP2 (Novus biologicals, Colorado, USA), mouse monoclonal anti-GFP (Roche, Basel, Switzerland), mouse monoclonal anti-γ-tubulin (GTU-88; Sigma-Aldrich), anti-human centromere protein (CREST patient serum; Antibodies Inc, California, USA), anti-KIF2A rabbit polyclonal (Novus biologicals), anti-Mad2 rabbit polyclonal (Bethyl Laboratories, Texas, USA), rabbit monoclonal (for ) and mouse monoclonal ( ) anti-phospho histone H3 (abcam, Cambridge, UK), rabbit monoclonal anti-HAUS6 (abcam), mouse anti-Human BUBR1 (BD Transduction Laboratories™, California, USA), and rabbit polyclonal anti-CAMSAP1 (Novus biologicals).

Techniques: Staining, Fluorescence, Control

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Scores of the signature genes resulted from the differential expression analysis using as input the EGA RNA-seq data compared to the three lung samples.

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Expressing

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: A The blot of siRNA BAG2 and MDK, C the blot of siRNA MAD2L1. β-Tubulin was used as a loading control and GAPDH was the positive control of the siRNA transfection. As shown in the blots and also in graphs ( B ) and ( D ) there was a significant reduction of the protein level in the silenced cells, as compared to the negative CTRL. E Graph showing the comparison of protein expression in MeT-5A and MSTO cell lines of BAG2, MAD2L1, and MDK (Mann–Whitney test; p value < 0.05 were considered significant, p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****)).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Control, Positive Control, Transfection, Comparison, Expressing, MANN-WHITNEY

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: A AOC of MeT-5A and MSTO at 72 h after treatment with siRNA-negative-control (siRNA CTRL−), siRNA BAG2, siRNA MAD2L1, siRNA MDK. The AOC is normalized to the time of transfection ( t 0 ). B Overall, FI of MeT-5A and MSTO was measured at 72 h after silencing; the graphs show the comparison between MeT-5A and MSTO for the three-siRNA transfection. ANOVA test; p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****). C – E End point of apoptosis analysis in MSTO and MeT-5A cells in the presence of 5 µM caspase 3/7 Green Dye and siRNA BAG2, siRNA MAD2L1, and siRNA MDK, respectively. Mann–Whitney test; p value 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Negative Control, Transfection, Comparison, MANN-WHITNEY

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: A – D Representative BAG2 immunostainings in RMP, EMM, BMM, and SMM, respectively. Moderate to strong expression in the three types of MPM, while no expression is visible in the hyperplastic mesothelium in RMP (red arrow). E – H Representative MAD2L1 immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is present in the three types of MPM, but there are areas of discernable expression in the mesothelial cells on the pleural surface in RMP as well (blue arrow positive mesothelial area, red arrow negative area). I – L Representative MDK immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is seen in the three types of MPM; however, some focal expression is also detectable in the mesothelial cells on the pleural surface in RMP (blue arrow focal staining, red arrow negative staining). Magnification: RMPs ×400, MPMs ×200.

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Expressing, Staining, Negative Staining

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Graphs for BAG2 immunostaining in RMP vs MPM regardless of subtype ( A ) and RMP vs the different subtypes of MPM ( B ), respectively. Graphs for MAD2L1 immunostaining in RMP vs MPM regardless of subtype ( C ) and RMP vs the different subtypes of MPM ( D ), respectively. Graphs for MDK immunostaining in RMP vs MPM regardless of subtype ( E ) and RMP vs the different subtypes of MPM ( F ), respectively. Kruskal–Wallis test followed by a post hoc Dunn’s test with Bonferroni correction was used to evaluate the statistical difference between the median of each group, p value <0.001(***), <0.01(**), <0.05(*).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Immunostaining

Journal: Cancer gene therapy

Article Title: BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma.

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Fig. 3 A representative western blot of the siRNA transfection experiment. A The blot of siRNA BAG2 and MDK, C the blot of siRNA MAD2L1. β-Tubulin was used as a loading control and GAPDH was the positive control of the siRNA transfection. As shown in the blots and also in graphs (B) and (D) there was a significant reduction of the protein level in the silenced cells, as compared to the negative CTRL. E Graph showing the comparison of protein expression in MeT-5A and MSTO cell lines of BAG2, MAD2L1, and MDK (Mann–Whitney test; p value < 0.05 were considered significant, p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****)).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant AntiBAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and AntiMAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Western Blot, Transfection, Control, Positive Control, Comparison, Expressing, MANN-WHITNEY

Journal: Cancer gene therapy

Article Title: BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma.

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Fig. 4 Viability, proliferation and caspase assays after gene silencing. A AOC of MeT-5A and MSTO at 72 h after treatment with siRNA- negative-control (siRNA CTRL−), siRNA BAG2, siRNA MAD2L1, siRNA MDK. The AOC is normalized to the time of transfection (t0). B Overall, FI of MeT-5A and MSTO was measured at 72 h after silencing; the graphs show the comparison between MeT-5A and MSTO for the three-siRNA transfection. ANOVA test; p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****). C–E End point of apoptosis analysis in MSTO and MeT-5A cells in the presence of 5 µM caspase 3/7 Green Dye and siRNA BAG2, siRNA MAD2L1, and siRNA MDK, respectively. Mann–Whitney test; p value 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant AntiBAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and AntiMAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Negative Control, Transfection, Comparison, MANN-WHITNEY

Journal: Cancer gene therapy

Article Title: BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma.

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Fig. 7 Representative immunohistochemical staining of MPM samples showing intense expression of MDK, MAD2L1, and BAG2 proteins in tumor cells (brown staining). A–D Representative BAG2 immunostainings in RMP, EMM, BMM, and SMM, respectively. Moderate to strong expression in the three types of MPM, while no expression is visible in the hyperplastic mesothelium in RMP (red arrow). E–H Representative MAD2L1 immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is present in the three types of MPM, but there are areas of discernable expression in the mesothelial cells on the pleural surface in RMP as well (blue arrow positive mesothelial area, red arrow negative area). I–L Representative MDK immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is seen in the three types of MPM; however, some focal expression is also detectable in the mesothelial cells on the pleural surface in RMP (blue arrow focal staining, red arrow negative staining). Magnification: RMPs ×400, MPMs ×200.

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant AntiBAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and AntiMAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, Negative Staining

Journal: Cancer gene therapy

Article Title: BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma.

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Fig. 8 Quantification of protein expression in RMP vs MPM as assessed by IHC and H-score. Graphs for BAG2 immunostaining in RMP vs MPM regardless of subtype (A) and RMP vs the different subtypes of MPM (B), respectively. Graphs for MAD2L1 immunostaining in RMP vs MPM regardless of subtype (C) and RMP vs the different subtypes of MPM (D), respectively. Graphs for MDK immunostaining in RMP vs MPM regardless of subtype (E) and RMP vs the different subtypes of MPM (F), respectively. Kruskal–Wallis test followed by a post hoc Dunn’s test with Bonferroni correction was used to evaluate the statistical difference between the median of each group, p value <0.001(***), <0.01(**), <0.05(*).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant AntiBAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and AntiMAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Expressing, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Coupling of Cdc20 inhibition and activation by BubR1

doi: 10.1083/jcb.202012081

Figure Lengend Snippet: Cdk1 phoshorylation of Cdc20 is required for an efficient mitotic arrest in response to microtubule poisons. (A) Live cell imaging of Hela cells depleted of endogenous Cdc20 and transiently expressing untagged Cdc20 WT, 3DD, or 3AA in the presence of nocadazole (30 ng/ml). Percentage of cells that exit mitosis during the course of imaging from three independent experiments (mean and SD indicated). (B) As in A, time from nuclear envelope breakdown (NEBD) to mitotic exit or end of filming from three independent experiments (each dot represents a single cell, red line indicates mean and SD, n values are shown in the graph and represent single cells analyzed per condition; statistical analysis with Mann-Whitney test; ****P > 0.0001). (C) Live cell imaging of stable cell lines that were depleted of endogenous Cdc20 and expressing indicated YFP-tagged Cdc20 proteins in the presence of nocodazole (30 ng/ml). The R132A mutation prevents Mad2 binding, while the 4A mutations prevent BubR1 binding. Time from NEBD to mitotic exit (each dot represents a single cell, red line indicates mean and standard deviation, n values are shown in the graph and represent single cells analyzed per condition, statistical analysis with Mann-Whitney test; ****P > 0.0001). (D) Model: MCC-bound BubR1 is preferentially dephosphorylated by PP2A-B56, whereas free Cdc20 is phosphorylated by Cdk1.

Article Snippet: The following antibodies were used at the indicated dilutions for Western blot: Cdc20 mouse monoclonal antibody (1:1,000; sc-13162; Santa Cruz Biotechnology; clone E7, indicated as #1), Cdc20 mouse monoclonal antibody (MAB3775; Millipore; clone AR12, indicated as #2), Cdc20 T70 and T59 phosphorylation-specific antibodies rabbit polyclonal antibody [raised against peptides CSKVQT(Tp)PSKPG and CRTPGR(Tp)PGKSS; Moravian Biotechnology; reference 22], APC3 T447p rabbit polyclonal [raised against Peptides CGKISTI(Tp)PQIQAF; Moravian Biotechnology], anti-phospho-histone H3 (Ser 10) rabbit polyclonal antibody (1:1,000; 06–570; Millipore), APC4 mouse monoclonal antibody (1:500; Moravian Biotechnology; clone CIV 1.1), APC3 mouse monoclonal antibody (1:500; Moravian Biotechnology), Mad2 rabbit polyclonal antibody (1:500; A300-300A; Bethyl Laboratories), Bub3 mouse monoclonal antibody (1:500; 611731; BD Biosciences), BubR1 rabbit polyclonal antibody (1:1,000; A300-995A; Bethyl Laboratories), and BubR1 mouse monoclonal antibody (1:500; Biotech Research and Innovation Centers; raised against TPR domain).

Techniques: Live Cell Imaging, Expressing, Imaging, MANN-WHITNEY, Stable Transfection, Mutagenesis, Binding Assay, Standard Deviation

Journal: Developmental cell

Article Title: The G2-to-M transition is ensured by a dual mechanism that protects cyclin B from degradation by Cdc20-activated APC/C

doi: 10.1016/j.devcel.2019.09.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Mad2 , Bethyl Laboratories , Cat #A300–301A; RRID AB_2281536.

Techniques: Virus, Recombinant, CRISPR, Software

Journal: Nature cell biology

Article Title: Ubiquitination-dependent localization of Polo-like kinase 1 in mitosis

doi: 10.1038/ncb2695

Figure Lengend Snippet: a, HeLa cells expressing H2B-mRFP were treated with the indicated siRNAs for 48 h and analyzed by live-cell microscopy. Stars indicate misaligned and lagging chromosomes. Bar is 10μm. b, The percentage of cells that complete mitosis normally or with a delay from prophase to anaphase (>50 min) or that undergo apoptosis during prometaphase was quantified (n = 50). c, The percentage of cells that undergo normal mitosis, become multinucleated or die during mitosis was quantified (n=50). d, HeLa cells were treated with the indicated siRNAs for 48 h and analyzed by immunofluorescence microscopy for MAD2 and kinetochores. The percentage of prometaphase and metaphase cells that contain one or more MAD2 positive kinetochores was quantified (n = 30). Example images are shown in Suppl. Fig. 1c. e, HeLa cells expressing H2B-mRFP were treated with the indicated mixtures of siRNAs for 48 h and analyzed by live-cell microscopy. The percentage of cells that die in prometaphase was quantified (n = 50). b-d, Bars represent the mean of three independent experiments. Error bars indicate +/-s.d.

Article Snippet: The following antibodies were used in the study: mouse monoclonal BubR1 (BD Biosciences 612502, 1:1000), pSer676 BubR1 (kind gift of E. A. Nigg, University of Basel, 1:???), CREST (Antibodies Incorporated, 15-234, 1:250), rabbit polyclonal CUL3 21 , mouse monoclonal FLAG (Sigma Aldrich F3165, 1:3000), mouse monoclonal GST (Labforce B-14, 1 :4000), rabbit polyclonal HA (Covance HA.11, 1 :3000), rabbit polyclonal KLHL22 and KLHL21 22 , rabbit polyclonal KLHL9 and 13 21 , rabbit polyclonal Mad2 (Bethyl Laboratories A300-301A, 1 :5000), mouse monoclonal MBP (Abcam ab49923, 1 :1000), rabbit polyclonal PLK1 (Abcam ab70697, 1:1000), mouse monoclonal PLK1 (Abcam ab17057, 1:500), rabbit polyclonal PLK2 (kind gift of I. Hoffmann, DKFZ Heidelberg, Germany), mouse monoclonal αTubulin (Sigma Aldrich T5169, IF 1:5000, WB 1:10000), rabbit polyclonal γTubulin (Sigma T3559, 1:1000), mouse monoclonal Cyclin A (Sigma C 4710, 1 :2000), mouse monoclonal Cyclin B1 (Santa Cruz GSN1, 1:5000), rabbit polyclonal phospho HistoneH3 (Upstate 06-570, 1:500), mouse monoclonal AuroraB (BD Biosciences 611082/3, 1:250), mouse monoclonal Hec1 (GeneTex, ??

Techniques: Expressing, Microscopy, Immunofluorescence