rabbit polyclonal anti aurora b  (Proteintech)

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti- Aurora B/AIM1 , Cell Signaling Technology , Cat# 3094, RRID: AB_10695307.

Techniques: Recombinant, Gene Expression, RNA Sequencing, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RetSat stabilizes mitotic chromosome segregation in pluripotent stem cells

doi: 10.1007/s00018-024-05413-x

Figure Lengend Snippet: RatSat localizes onto mitotic chromosomes in pluripotent stem cells. a-b : Representative images ( a ) and quantification ( b ) of immunostaining assay using anti-RetSat antibody in designated stemness positive (mESCs, hESCs, miPSCs) and negative (MEF, HEL-1, 293T) cell lines. Aurora B was used as a mitotic marker. Chromosomes were labeled by DAPI. Scale bar represents 5 μm. c . Neuronal lineage differentiation of mouse embryonic stem cells using LIF-free, RA-plus procedure. d-e : Confirmation of differentiation efficiency using qPCR ( d ) and immunoblotting ( e ). Gapdh was used as an immunoblotting loading control. f-g . Percentage ( f ) and representative images ( g ) using anti-RetSat antibody in mitotic mESCs and isogenic differentiation counterparts. n = 50 in each group. Scale bar represents 5 μm. h . Pluripotency reprogramming of mouse embryonic fibroblasts using Yamanaka factors transduction procedure. i-j : Confirmation of pluripotency reprogramming efficiency using qPCR ( i ) and immunoblotting ( j ). β-actin was used as an immunoblotting loading control. k-l : Percentage ( k ) and representative images ( l ) using anti-RetSat antibody in mitotic MEFs and isogenic reprogramming counterparts. n = 50 in each group. Scale bar represents 5 μm

Article Snippet: The following antibodies were obtained from the indicated suppliers: rabbit anti-RetSat polyclonal antibody (Invitrogen, Cat. No. PA5-65443, 1:1000 for immunoblotting), mouse anti-RetSat monoclonal antibody (made in our laboratory, we have isolated and purified a high concentration of single epitope specific RetSat monoclonal antibody at a concentration of ∼ 0.8 mg/ml after fusion culture and screening of hybridoma cells from mice immunized with RetSat antigen, 1:400 for immunoblotting, 1:20 for immunofluorescence and 3 µg for immunoprecipitation), rabbit anti-Aurora B polyclonal antibody (Abcam, Cat. No. ab2254, 1:500 for immunofluorescence), rabbit anti-Oct4 monoclonal antibody (Abcam, Cat. No. ab181557, 1:1000 for immunoblotting), rabbit anti-Sox2 monoclonal antibody (Abcam, Cat. No. ab92494, 1:1000 for immunoblotting), rabbit anti-Nudcd2 polyclonal antibody (Proteintech, Cat. No. 21205-1-AP, 1:1000 for immunoblotting), mouse anti-Smc1a monoclonal antibody (Proteintech, Cat. No. 66987-1-Ig, 1:1000 for immunoblotting), rabbit anti-Smc3 polyclonal antibody (Proteintech, Cat. No. 14185-1-AP, 1:500 for immunoblotting), mouse anti-Gapdh monoclonal antibody (Beyotime, Cat. No. AF0006, 1:1000 for immunoblotting), mouse anti-β-Actin monoclonal antibody (Sigma-Aldrich, Cat. No. A1978, 1:2000 for immunoblotting), rabbit anti-Histone H2A polyclonal antibody (Beyotime, Cat. No. AH419, 1:1000 for immunoblotting), mouse IgG antibody (Beyotime, Cat. No. A7028, 1 µg for immunoprecipitation).

Techniques: Immunostaining, Marker, Labeling, Western Blot, Control, Transduction

Journal: PLoS ONE

Article Title: Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

doi: 10.1371/journal.pone.0028011

Figure Lengend Snippet: A. U937 cells were transfected with control DNA, IER5 cDNA, or were treated with TMPP (5 µM or 10 µM). The cells were harvested after 3 days, and the expression of the indicated cell cycle regulating proteins was analyzed by SDS-PAGE followed by Western blotting using the indicated specific antibodies. Blotting of Actin was used as a loading control. B. Schematic representation of the human Cdc25B promoter regions, indicating sites for Sp1, Sp3, and NF-YB motifs, amplified from the precipitated DNA by specific primer sets 1 and 2. C. ChIP analysis for IER5 in the Cdc25B promoter. D & E. IER5-dependent changes in Sp1, Sp3 and NF-YB binding (E), and the bindings of coactivators, DNMT1 and p300, to interact with Sp1 and NF-YB, respectively (F) in Cdc25B promoter. U937 cells were either untransfected or were transfected with the control DNA or IER5 DNA. After 3 days culture, cells were cross-linked with 1% formaldehyde and ChIP was performed with either control antibody (IgG) or the anti-IER5 antibody. The precipitated DNA was then assayed by PCR using pairs (primer set 1 and 2) of oligonucleotides encompassing specific regions of the Cdc25B promoter. The values of ChIP efficiencies are given as % of input.

Article Snippet: Western blot analysis was performed using the following antibodies: goat polyclonal anti-IER5 (Abcam, Cambridge, UK); Rabbit polyclonal anti-Cdc25B, anti-CHK1, anti-WEE1 and anti-Aurora-B; mouse monoclonal anti-Cyclin B1 and anti-Survivin, all from Santa Cruz.

Techniques: Transfection, Expressing, SDS Page, Western Blot, Amplification, Binding Assay

Journal: PLoS ONE

Article Title: Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

doi: 10.1371/journal.pone.0028011

Figure Lengend Snippet: A. Selection of ALDH hi /CD34 + hematopoietic progenitor cells from the bone marrow of two healthy volunteers (#1 and #2) by FACS sorting. Region P denotes populations of ALDH hi cells. Region R and S denote populations of CD34 + and CD34 - cells in the ALDH hi population (Region P), respectively. Negative control, light grey region; CD34-PE staining, dark grey region. B. Selection of ALDH hi /CD34 + hematopoietic progenitor cells from the bone marrow of two AML patients (M1 and M2) by FACS sorting. Region P denotes populations of ALDH hi cells. Region R and S denote populations CD34 + and CD34 - cells in the ALDH hi population (Region P), respectively. Negative control, light grey region; CD34-PE staining, dark grey region. C. ALDH hi /CD34 + cells were purified from a healthy volunteer (#1) and an AML patient (M1), and were cultured in semisolid methylcellulose media. The ALDH hi /CD34 + cells from each source were left untransfected or were transfected with IER5 cDNA, or treated with TMPP (5 µM). After 14 days culture, the colony forming ability of the cells was analyzed (left upper panel) and the cells were viewed using phase-contrast microscopy. Original magnification ×4 (left bottom panels). Their mRNA expression of IER5 and Cdc25B was assessed using RT-PCR and quantitative RT-PCR (right panels). Colonies formed by these ALDH hi /CD34 + cells (3×10 2 to 5×10 2 cells/plate) were counted following plating in semisolid methylcellulose media. Colony formation was evaluated by determination of colony counts as a percentage of the corresponding control. The results are the means ± SD of three independent experiments. * P <0.01 compared with untreated control cells. RT-PCR results representative of three independent experiments are shown. GAPDH mRNA expression is shown as an internal control. The ALDH hi /CD34 + cells whose IER5 mRNA expression was analyzed by quantitative RT-PCR were derived from an AML patient (M1). The levels of the quantified RT-PCR products were normalized t o GAPDH expression in the same sample and were then expressed relative to the mRNA level of a normal control which was assigned a value of 1. D and E. Cell cycle analysis (D) and changes of mitochondrial membrane potential (ΔΨm) (E) in IER5 overexpressed or TMPP treated AML-derived ALDH hi /CD34 + cells. ALDH hi /CD34 + cells were purified from an AML patient (M1), and were then transfected with IER5 cDNA or were treated with TMPP (5 µM). The IER5-transfected or TMPP-treated cells were harvested after 3 days. The cell cycle distribution and the ΔΨm of the ALDH hi /CD34 + cells was analyzed using flow cytometric analysis. The FACS results are representative of three independent experiments. NC; Negative control.

Article Snippet: Western blot analysis was performed using the following antibodies: goat polyclonal anti-IER5 (Abcam, Cambridge, UK); Rabbit polyclonal anti-Cdc25B, anti-CHK1, anti-WEE1 and anti-Aurora-B; mouse monoclonal anti-Cyclin B1 and anti-Survivin, all from Santa Cruz.

Techniques: Selection, Negative Control, Staining, Purification, Cell Culture, Transfection, Microscopy, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Cell Cycle Assay

Journal: PLoS ONE

Article Title: Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

doi: 10.1371/journal.pone.0028011

Figure Lengend Snippet: A. ALDH hi /CD34 + cells were purified from an AML patient (M1) and were cultured in semisolid methylcellulose media. The ALDH hi /CD34 + cells were either untransfected or were transfected with IER5 cDNA or with shRNA-#1, or -#2. After 14 days culture with or without TMPP (5 µM), the cells were then analyzed for their colony forming ability. Colony formation of these ALDH hi /CD34 + cells (3×10 2 to 5×10 2 cells/plate) was assessed after plating in semisolid methylcellulose media. B. Analysis of the mRNA expression of IER5 and Cdc25B in each colony using QRT-PCR and RT-PCR. C. The cells were viewed using phase-contrast microscopy after 14 days culture with or without TMPP (5 µM). Original magnification ×4. D. ALDH hi /CD34 + cells were purified from an AML patient (M1) and were cultured in semisolid methylcellulose media. The ALDH hi /CD34 + cells were either untransfected or were transfected with IER5 cDNA or Cdc25B cDNA. After 14 days culture with or without TMPP (5 µM), the cells were then analyzed for their colony forming ability. Colony formation of these ALDH hi /CD34 + cells (3×10 2 to 5×10 2 cells/plate) was assessed after plating in semisolid methylcellulose media. Colony formation was evaluated as a percentage of the corresponding control. E. Analysis of the mRNA expression of IER5 and Cdc25B in each colony using QRT-PCR and RT-PCR. The levels of the QRT-PCR products were normalized t o GAPDH expression in the same sample and were then expressed relative to the mRNA level of a normal control which was assigned a value of 1. RT-PCR results representative of three independent experiments are shown. GAPDH mRNA expression is shown as an internal control. The results are the means ± SD of three independent experiments. * P <0.01 compared with untreated control cells. F. The cells transfected with Cdc25B or IER5 cDNA were viewed using phase-contrast microscopy after 14 days culture with or without TMPP (5 µM). Original magnification ×4.

Article Snippet: Western blot analysis was performed using the following antibodies: goat polyclonal anti-IER5 (Abcam, Cambridge, UK); Rabbit polyclonal anti-Cdc25B, anti-CHK1, anti-WEE1 and anti-Aurora-B; mouse monoclonal anti-Cyclin B1 and anti-Survivin, all from Santa Cruz.

Techniques: Purification, Cell Culture, Transfection, shRNA, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Microscopy

Journal: PLoS ONE

Article Title: Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

doi: 10.1371/journal.pone.0028011

Figure Lengend Snippet: The ALDH hi /CD34 + cells purified from two AML patients (AML: M1) were either untransfected or were transfected with control DNA or IER5 DNA. After 14 days culture, cells were cross-linked with 1% formaldehyde and ChIP was performed with either control antibody (IgG) or the anti-IER5 antibody. The precipitated DNA was then assayed by real-time PCR using pairs (primer set 2) of oligonucleotides encompassing specific region of the Cdc25B promoter (left panel). IER5-dependent changes in NF-YB binding (middle panel), and the bindings of coactivators, p300 (right panel) in Cdc25B promoter. The values of ChIP efficiencies are given as % of input.

Article Snippet: Western blot analysis was performed using the following antibodies: goat polyclonal anti-IER5 (Abcam, Cambridge, UK); Rabbit polyclonal anti-Cdc25B, anti-CHK1, anti-WEE1 and anti-Aurora-B; mouse monoclonal anti-Cyclin B1 and anti-Survivin, all from Santa Cruz.

Techniques: Purification, Transfection, Real-time Polymerase Chain Reaction, Binding Assay

Journal: PLoS ONE

Article Title: Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/C Cdh1 Complex

doi: 10.1371/journal.pone.0057532

Figure Lengend Snippet: (A) Normalized expression levels of the mouse genes GAS2, GAS2L1, GAS2L3, and CCNA2 in naïve, plasmablast, germinal center (GC), and memory-B cells. Raw data were taken from the GEO database GSE4142. (B) Real-time PCR analysis of human GAS2L3 and CCNA2 expression throughout the cell cycle of HeLa S3 (S3) cells, normalized to ACTB RNA levels at time point 0. Cells were double-thymidine blocked, released, and harvested for RNA extraction every 2 hrs for 24 hrs. Cell cycle progression of the synchronous populations (measured by propidium-iodide (PI) staining followed by FACS analysis) is depicted under the plot. (C) HEK293 (293) cells overexpressing human Gas2l3 or Gas2l3-EGFP were harvested for Western blot analysis with custom-made polyclonal hGas2l3 antibodies (serum). A cross-reactive band is noted (asterisk) in the transfected and untransfected (UT) cells. Forty µg extracts made from transfected cells and 120 µg extracts made from untransfected cells were assayed. (D) Left: 293 cells were transfected with the Gas2l3 expression vector. After 24 hrs, cells were treated with MG132 for 5 hrs and subjected to Western blotting with Gas2l3 (serum) and Actin antibodies (loading control). Right: Human Gas2l3 was expressed in rabbit reticulocytes supplemented with radiolabeled ( 35 S) methionine. In vitro transcribed/translated (IVT) Gas2l3 product was incubated in G1 extracts of S3 cells in the presence of MG132 or DMSO (control). Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (E) 35 S-labeled hGeminin, hTome-1, and hGas2l3 IVT products were incubated in G1-phase S3 cell extracts supplemented with either buffer, the C-terminus of hEmi1, or hSecurin. Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (F) Left: The putative D-boxes of human Gas2l3 are depicted. Right: Arg at position 1 and Leu at position 4 of each putative D-box were substituted with Gly and Val, respectively. The degradation of the four D-box mutants (DM1–DM4) was assayed as described in (D). (G) 293 cells were cotransfected with Cdh1 (+) or empty vector (-), and with either Gas2l3 or its mutant derivate Gas2l3-DM4, at a 4∶1 ratio, respectively. After 30 hrs cells were harvested for Western blotting with anti-hGas2l3 (serum) and anti-Tubulin (control).

Article Snippet: The following primary antibodies were used for IF: rabbit polyclonal anti-hGas2l3 (serum, custom-made by Covance), mouse monoclonal and rabbit polyclonal anti-Aurora B (Abcam, ab3609, ab2254), rabbit polyclonal MKLP1 (Gene Tex, GTX30315), mouse monoclonal anti-αTubulin (Abcam, ab7291), mouse monoclonal anti-FLAG® M2 (Sigma-Aldrich, F3165), and mouse monoclonal anti-Myc (DSHB, 9E10).

Techniques: Expressing, Real-time Polymerase Chain Reaction, RNA Extraction, Staining, Western Blot, Transfection, Plasmid Preparation, In Vitro, Incubation, SDS Page, Autoradiography, Labeling, Mutagenesis

Journal: PLoS ONE

Article Title: Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/C Cdh1 Complex

doi: 10.1371/journal.pone.0057532

Figure Lengend Snippet: (A) Schematic representation of the midbody. (B) HeLa cells expressing EGFP-tagged Gas2l3 were fixed, immunolabeled with either anti-Aurora B or anti-Tubulin, and stained with DAPI. The localization of proteins to the midbody was determined using linescans (stembody and constriction sites are labeled by red and white arrows, respectively). (C) HeLa cells were fixed and immunolabeled with anti-hGas2l3 (serum), and with either anti-Aurora B or anti-Tubulin. Linescans for cells 1 and 2 are plotted. (D) HeLa cells were fixed and immunolabeled with anti-hGas2l3 (serum). The immunofluorescent intensity of the endogenous Gas2l3 at six points across one side of the midbody were measured relative to the width of the intercellular microtubule bridge at these locations (see average and standard error values for N = 5 at the plot, and the scheme underneath). Width and intensity values were normalized to the intercellular bridge width and Gas2l3 intensity at the constriction sites. (E) HeLa cells expressing FLAG-tagged human CHMP4b (top panels), Gas2l3-EGFP and FLAG-CHMP4b (mid panels), or Gas2l3-EGFP and Myc-tagged human Spastin (bottom panels), were fixed and immunolabeled with anti-hGas2l3 (serum [top panels]), anti-FLAG (mid panels), or anti-Myc (bottom panels). Linescans are depicted. We used a Zeiss AxioImager and 100X oil lens for imaging, and ImageJ software for image analysis.

Article Snippet: The following primary antibodies were used for IF: rabbit polyclonal anti-hGas2l3 (serum, custom-made by Covance), mouse monoclonal and rabbit polyclonal anti-Aurora B (Abcam, ab3609, ab2254), rabbit polyclonal MKLP1 (Gene Tex, GTX30315), mouse monoclonal anti-αTubulin (Abcam, ab7291), mouse monoclonal anti-FLAG® M2 (Sigma-Aldrich, F3165), and mouse monoclonal anti-Myc (DSHB, 9E10).

Techniques: Expressing, Immunolabeling, Staining, Labeling, Imaging, Software

Journal: PLoS ONE

Article Title: A RasGAP SH3 Peptide Aptamer Inhibits RasGAP-Aurora Interaction and Induces Caspase-Independent Tumor Cell Death

doi: 10.1371/journal.pone.0002902

Figure Lengend Snippet: (A) Silencing of Aurora A and B expression. HeLa cells were transfected with scrambled (Scr) siRNAs or with siRNAs directed against Aurora A or B (Cont: untransfected cells). Aurora A and B expression was revealed by Western blot experiments using specific antibodies, and γ-tubulin expression was used as a control (B) HeLa cells were co-transfected with a plasmid directing the expression of either control aptamer C6 or RG27, and with siRNAs directed against Aurora A, Aurora B, or Survivin (“Untreated”: no siRNA; “Scr”: scrambled siRNA). Cells were labeled and analyzed as in .

Article Snippet: We washed the beads three times in Hepes 50 mM pH7.5, NaCl 150 mM, Triton 0.1%, boiled them in sample buffer and ran a SDS-PAGE followed by a Western blot using the mAb200 RasGAP SH3 antibody (1∶1000) or a rabbit polyclonal Aurora B antibody (Abcam) (1∶2500).

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Labeling

Journal: PLoS ONE

Article Title: A RasGAP SH3 Peptide Aptamer Inhibits RasGAP-Aurora Interaction and Induces Caspase-Independent Tumor Cell Death

doi: 10.1371/journal.pone.0002902

Figure Lengend Snippet: (A,B,C) RasGAP/Aurora B co-capture assay. Upper panels: A RasGAP pulldown assay was performed from HeLa cells (A), HCT116 cells (B) and Panc 10.05 cells (C) expressing control aptamer C6 or RG27, using either a RasGAP-SH2-interacting peptide (DOK) coupled to CNBr-sepharose beads or uncoupled beads (Cont.). Captured RasGAP and Aurora B proteins were revealed by Western blotting. Lower panels: Western blot experiments were performed from a fraction of the total HeLa, HCT116, and Panc 10.05 cell lysates used in the pulldown assays, and from lysates of non-transfected cells (NT), using RasGAP and Aurora B specific antibodies. (D) Aurora B kinase activity. The content of phosphorylated Histone H3 was determined by Western blot from soluble protein extracts of HeLa cells expressing either C6 or RG27 peptide aptamers. A Western blot using a γ-tubulin antibody was performed to obtain a loading control. Histogram shows the quantification of three independent experiments.

Article Snippet: We washed the beads three times in Hepes 50 mM pH7.5, NaCl 150 mM, Triton 0.1%, boiled them in sample buffer and ran a SDS-PAGE followed by a Western blot using the mAb200 RasGAP SH3 antibody (1∶1000) or a rabbit polyclonal Aurora B antibody (Abcam) (1∶2500).

Techniques: Expressing, Western Blot, Transfection, Activity Assay