rabbit polyclonal anti phospho aurora a  (Cell Signaling Technology Inc)

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

Journal: iScience

Article Title: EMC3 regulates mesenchymal cell survival via control of the mitotic spindle assembly

doi: 10.1016/j.isci.2022.105667

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Phospho-Aurora A (Thr288) , Invitrogen , Cat#44–1210G LOT#RG241362; RRID: AB_1461184.

Techniques: Recombinant, Staining, In Situ, Extraction, Transfection, Flow Cytometry, Isolation, Mass Spectrometry, Control, shRNA, Software

Journal: iScience

Article Title: EMC3 regulates mesenchymal cell survival via control of the mitotic spindle assembly

doi: 10.1016/j.isci.2022.105667

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) , Cell signaling , Cat#2914S LOT#9; RRID: AB_2061631.

Techniques: Recombinant, Staining, In Situ, Extraction, Transfection, Flow Cytometry, cDNA Synthesis, Isolation, Mass Spectrometry, Control, shRNA, Software, Quantitative Proteomics