Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: Protein expression and subcellular localization of AURKA during meiotic maturation. (A) Immunoblot blot analysis of AURKA in cumulus-free oocytes (200 oocytes per lane) cultured in vitro to various stages—GV (0 h), GVBD (1 h), MI (7 h), MII (18 h). The amount of AURKA protein increased slightly around GVBD. (B) Quantification of immunoblots. The experiment was performed three times and the data are expressed as mean ±SEM. Statistical differences (p < 0.05) in comparison to GV-stage are marked (*) (C) Phase-contrast and fluorescence microscopy images of oocytes after co-injection of Gfp-Aurka mRNA (2–5 pl of 200 ng/μl) and mCherry-H2B (2–5 pl of 25 ng/μl) into GV-stage. Total AURKA was mainly present at MTOC at GV-stage oocytes, at MI and MII AURKA was mainly associated with spindle with a clear concentration on the spindle poles and cytoplasmic MTOCs (arrowheads).

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Expressing, Western Blot, Cell Culture, In Vitro, Comparison, Fluorescence, Microscopy, Injection, Concentration Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: PI3K-PKB and CDK1 independent AURKA activation precedes resumption of meiosis. Localization of pT288 AURKA (active form) during meiotic maturation (A). MTOC-associated AURKA is phosphorylated on T288 before GVBD (bGVBD), then during prometaphase I (PMI) and metaphase I (MI) pT288 AURKA remains associated with MTOCs. Green—pT288 AURKA, red—pericentrin (MTOCs). (B) Inhibition of CDK1 activity does not prevent activation of AURKA and multiplication of MTOCs. Oocytes, cultured for 90 min in the presence of Roscovitine, showed phosphorylation of AURKA on T288 at amplified MTOCs or within the nucleus. (C) Ability of LY-294002 or SH-6 oocytes to resume meiosis. The experiment was performed four times and around 200 oocytes were counted for each group. Error bars show confidence intervals. Statistical differences (p < 0.05) in comparison to control are marked (*) (D) PI3K-PKB signaling pathway is not involved in AURKA activation. Oocytes were cultured in medium supplemented with LY-294002 or SH-6 for 90 min and then GV-stage oocytes were used for imunofluorescence staining. Inhibition of PI3K or PKB did not block phosphorylation of AURKA, multiplication of MTOC and induced presence of pT288 AURKA within the nucleus.

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Activation Assay, Inhibition, Activity Assay, Cell Culture, Phospho-proteomics, Amplification, Comparison, Control, Staining, Blocking Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: Overexpression of AURKA triggers MTOCs multiplication in GV-arrested oocytes. Gfp-Aurka. mRNA was microinjected into GV-blocked oocytes (2–5 pl of 250 ng/μl); Egfp mRNA was used as a control. (A) Exogenous AURKA caused multiplication of MTOCs in the presence of an intact GV after overnight incubation. GFP-AURKA was located on all MTOCs, (B) but 2 h after injection was non-phosphorylated. Overnight incubation led to phosphorylation of exogenous GFP-AURKA on T288. Pericentrin was used as a MTOC marker. (C) Endogenous AURKA is not phosphorylated during overnight culture in IBMX-containing medium (D) Activity of CDK1 (arbitrary units) was assayed on extracts from 10 oocytes per sample. The experiment was done 3 times and activity is expressed as mean ± SEM. Statistical difference (p < 0.05) in comparisons to GV-stage control is marked (*).

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Over Expression, Control, Incubation, Injection, Phospho-proteomics, Marker, Activity Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: MI spindle defects induced by AURKA overexpression. (A) Aurka mRNA (2–5 pl, 350 ng/ul) injected oocytes, cultured for 18 h, were evaluated. Overexpression of AURKA blocks oocytes in PMI/MI. Altogether 100 GFP (control) and 99 GFP-AURKA oocytes were evaluated in three independent experiments. Error bars show confidence intervals. Statistical differences (p < 0.05) in individual cell cycle stages (MI and MII) are marked (*), difference in GV stage is not significant (B) Morphological defects such as formation of abnormally long spindle, absence of congression of tetrads or formation of unipolar (arrow) and multipolar spindles (arrowheads) were analyzed by immunocytochemistry. The 83 injected oocytes from three independent experiments were evaluated and morphologically analyzed. Red—acetylated α-tubulin, blue—DAPI.

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Over Expression, Injection, Cell Culture, Control, Immunocytochemistry

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: Downregulation of AURKA leads to incorrect spindle assembly and PMI/MI arrest. Oocytes injected with Aurka dsRNA or with Egfp dsRNA as a control were cultured for 24 h in IBMX-supplemented medium. (A) Aurka mRNA level in relative arbitrary units after RNAi mediated knockdown in GV-arrested oocytes. Total RNA was subsequently isolated and used for real-time PCR to quantify the level of Aurka mRNA. Egfp was used as an external standard. The decrease in the amount of Aurka mRNA was significant (p < 0.05). Data are expressed as the mean ± SEM. (B) Meiotic maturation of dsRNA injected oocytes. Oocytes after microinjection of Aurka dsRNA are arrested PMI/MI, when evaluated 18 h after IBMX release. 109 GFP RNAi and 94 AURKA RNAi oocytes were analyzed in three experiments. Statistical differences (p < 0.05) in individual cell cycle stages (GV, MI and MII) are marked (*) (C) Phenotypes of AURKA knockdown in mouse oocytes. Representative images of three experiments (approx. 100 oocytes) showing contol MI phase spindle of oocytes injected with Egfp (left) and oocytes injected with Aurka dsRNA (right) at the time 7 h after their transfer to IBMX-free medium. Red—acetylated α-tubulin, blue—DAPI for DNA staining.

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Injection, Control, Cell Culture, Knockdown, Isolation, Real-time Polymerase Chain Reaction, Microinjection, Staining

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Successive recruitment of p-CDC25B-Ser351 and p-cyclin B1-Ser123 to centrosomes contributes to the release of mouse oocytes from prophase I arrest.

doi: 10.1002/dvdy.24220

Figure Lengend Snippet: Fig. 6. Ser351 is a potential AURKA phosphorylation target of CDC25B. A: At indicated times after release, control siRNA injected oocytes were collected, fixed, and double stained with antibodies to AURKA (red) or phospho-CDC25B-Ser351 (green). B: At indicated times after release, con- trol siRNA injected oocytes were collected, fixed, and double stained with antibodies to phospho-AURKA-Thr288 (green) or pericentrin (red). The fluorescence profiles show the intensity and association between two stained proteins. Scale bars ¼ 20 mm.

Article Snippet: Blocked oocytes were incubated overnight at 4 C with a rabbit anti-phospho-Cyclin B1-Ser126 polyclonal antibody diluted 1:500 (Abcam); rabbit anti-phospho-CDC25B-Ser351 polyclonal antibody diluted 1:400 (Signalway Antibody); rabbit antipericentrin polyclonal antibody diluted 1:400 (Abcam); mouse anti-AURKA monoclonal antibody (mAb) diluted 1:100 (Abcam); rabbit anti-phospho-AURKA-Thr288 mAb diluted 1:500 (Cell Signaling, Danvers, MA); mouse anti-pericentrin mAb diluted 1:50 (BD Transduction Laboratories, San Diego, CA); mouse antia-tubulin mAb diluted 1:100 (Life Technologies, Carlsbad, CA) and mouse anti-lamin A/C mAb diluted 1:50 (Cell Signaling) at 4 C. After being washed 3 5 min in PBS with 0.3% BSA, the oocytes were incubated with FITC-conjugated goat anti-rabbit secondary antibody (1:200 dilution in blocking solution) and TRITC-conjugated goat anti-mouse secondary antibody (1:200 dilution in blocking solution) at room temperature for 1 hr.

Techniques: Phospho-proteomics, Control, Injection, Staining, Fluorescence

Journal: Molecular Therapy

Article Title: LINC00470 Coordinates the Epigenetic Regulation of ELFN2 to Distract GBM Cell Autophagy

doi: 10.1016/j.ymthe.2018.06.019

Figure Lengend Snippet: ELFN2 Binds to AurkA and eIF2α and Upregulates eIF2α by Regulating the Kinase Activity of AurkA (A) Representative confocal and immunofluorescence images showing the co-localization of ELFN2 (red) and AurkA (green) in HEK293 cells. (B) coIP analysis showing the interaction between ELFN2 and AurkA in HEK293 and U251 cells. (C) coIP analysis showing the interaction between ELFN2 and AurkA domains in HEK293 cells. (D) Representative confocal and immunofluorescence images showing the co-localization of ELFN2 (red) and eIF2α (green) in HEK293 cells. (E) coIP analysis showing the interaction between ELFN2 and eIF2α in HEK293 cells. (F) Representative confocal and immunofluorescence images showing the co-localization of AurkA (red) and eIF2α (green) in HEK293 cells. (G) coIP analysis showing the interaction between AurkA and eIF2α in HEK293 cells. (H) GST pull-down assays showed that the reg2 domain of AurkA pulled down ELFN2. (I) GST pull-down assays showed that the reg1 and reg2 domains of AurkA pulled down eIF2α. (J) qRT-PCR was performed to detect the expression of eIF2α in GBM cells after the ELFN2 knockdown. The data are presented as the means ± SEM of three independent experiments. *p < 0.05. (K) Western blotting was performed to detect the levels of AurkA and p-AurkA in GBM cells transfected with si-ELFN2. (L) qRT-PCR was performed to detect the expression of eIF2α in GBM cells after ELFN2 knockdown and AurkA overexpression. The data are presented as the means ± SEM of three independent experiments. **p < 0.01. (M) Western blotting was performed to detect the level of autophagy markers in ELFN2-overexpessing GBM cells after AurkA or eIF2α knockdown.

Article Snippet: The reagents, chemicals, and antibodies used in this study are as follows: LC3 (Cell Signaling Technology, 12741), Beclin-1 (Cell Signaling Technology, 3495), Atg5 (Cell Signaling Technology, 12994), Atg12 (Cell Signaling Technology, 4180), Atg7 (Cell Signaling Technology, 8558), Atg3 (Cell Signaling Technology, 3415), GFP (Proteintech, 50430-2-AP), ELFN2 (Sigma, HPA00781), phospho-AurkA (Cell Signaling Technology), AurkA (Cell Signaling Technology, 14475), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Proteintech, 60004-1-Ig), FLAG (Sigma-Aldrich, F3165), and rapamycin (MedChemExpress, HY-10219).

Techniques: Activity Assay, Immunofluorescence, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Over Expression

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: eBioMedicine

Article Title: The ubiquitin-ligase TRAF6 and TGFβ type I receptor form a complex with Aurora kinase B contributing to mitotic progression and cytokinesis in cancer cells

doi: 10.1016/j.ebiom.2022.104155

Figure Lengend Snippet: APPL1 and 2 promote AURKB, BIRC5, CDCA8 , and KIF2C expression. (a) Human prostate cancer PC-3U cells were transfected with control or No. 1 APPL1 and APPL2 siRNA. RNA was extracted from cells, and microarray analysis was performed. (b) qRT-PCR analysis of the genes shown in panel a of cells treated with or without No. 1 APPL1 and APPL2 siRNA. Inhibition by siRNA was overcome by expressing siRNA-resistant constructs; N = 4, data presented as mean±SEM [Student's t-test, * p < 0.05, ** p < 0.01, *** p < 0.001]. (c) PC-3U cells were synchronized with a double thymidine block and treated with No. 1 APPL1 and APPL2 siRNA. Cells were released and cell lysates were prepared at different times, and subjected to immunoblotting. (d) PC-3U cells were transfected with or without No. 1 APPL1 and APPL2 siRNA, incubated with nocodazole for 12 h, and analyzed by immunoblotting. (e) Immunofluorescence and confocal imaging showing co-localization of AURKB (green) and APPL1 (red) during telophase and cytokinesis. (f-k) Orthogonal views (XY, XZ and YZ) of two Z-stack images of panel e. (f, i) XY view (z-projection). (g, j) XZ view. (h, k) YZ view. Scale bar, 20 µm. (l) Schematic representation of the APPL1 protein and mutants. (m) PC-3U cells transiently transfected with HA-AURKB and different APPL1 domains as indicated, were synchronized and then subjected to immunoprecipitation with an antibody against HA and immunoblotting using a GFP antibody. Non-transfected (NT).

Article Snippet: Other primary antibodies against the following proteins were used in immunofluorescence experiments: AURKB (Novus, Cat# NBP2-50039, RRID:AB_2895237), and p-Smad2 (Cell Signaling Technology Cat# 3108, RRID:AB_490941).

Techniques: Expressing, Transfection, Microarray, Quantitative RT-PCR, Inhibition, Construct, Blocking Assay, Western Blot, Incubation, Immunofluorescence, Imaging, Immunoprecipitation

Journal: eBioMedicine

Article Title: The ubiquitin-ligase TRAF6 and TGFβ type I receptor form a complex with Aurora kinase B contributing to mitotic progression and cytokinesis in cancer cells

doi: 10.1016/j.ebiom.2022.104155

Figure Lengend Snippet: TβRI co-localizes with AURKB during mitosis. (a-c) Immunofluorescence experiments showing co-localization of AURKB (green) and TβRI (V22, red) during mitosis in human prostate cancer (PC-3U) (a) and human neuroblastoma (KELLY) (b) cells, and of TβRI (V22, green) and β-tubulin (red) throughout the PC-3U mitosis (c). Scale bar, 20 µm. (d) Decreased co-localization of TβRI and AURKB after treatment of PC-3U cells on ice for 30 min. Scale bar, 20 µm. (e) Multinucleated cells were counted after knockdown of TGFBR1 . Data presented as mean±SEM, N=3 [Student's t-test, * p < 0.05]. Scale bar, 20 µm. (f) Gene Set Enrichment Analysis (GSEA) of genes ranked by their correlation with TGFBR1 expression yielded 34 significantly enriched gene sets (adjusted p -value ≤ 0.05 and the p -values are adjusted using the Benjamini-Hochberg procedure). The ridge-plot shows the distribution of correlation coefficients of the core enriched genes, i.e., genes which contribute most to the enrichment of the gene set. The gene sets are ordered by normalized enrichment score. Color indicates the adjusted p -value. (g) GSEA plots of the hallmark mitotic spindle (left) and G2/M checkpoint (right) gene sets show their strong association with TGFBR1 -correlated genes. The upper panels show the correlation coefficients and position of the gene set genes within the ranked list of all genes, and the lower panels show the running enrichment score.

Article Snippet: Other primary antibodies against the following proteins were used in immunofluorescence experiments: AURKB (Novus, Cat# NBP2-50039, RRID:AB_2895237), and p-Smad2 (Cell Signaling Technology Cat# 3108, RRID:AB_490941).

Techniques: Immunofluorescence, Expressing

Journal: eBioMedicine

Article Title: The ubiquitin-ligase TRAF6 and TGFβ type I receptor form a complex with Aurora kinase B contributing to mitotic progression and cytokinesis in cancer cells

doi: 10.1016/j.ebiom.2022.104155

Figure Lengend Snippet: TRAF6 mediates K63-linked polyubiquitination of AURKB. (a-b) PC-3U cells were treated with or without TRAF6 siRNA, synchronized with a double thymidine block and subjected to analysis by immunoblotting (IB) after different time periods (a), with or without incubation for 12 h with nocodazole (b). (c) Lysates of synchronized PC-3U cells were immunoprecipitated (IP) with an AURKB antibody, followed by immunoblotting with antibodies against TβRI, APPL1 and TRAF6. (d) Lysates of synchronized PC-3U cells transfected with Flag-AURKB and HA-tagged wild-type (WT) or mutated ubiquitin, were subjected to immunoprecipitation using a Flag antibody, followed by immunoblotting using an HA antibody. Arrow points to heavy immunoglobulin chain. (e) PC-3U cells were synchronized with a double thymidine block, released to fresh media with 10% FBS, harvested at the indicated times, and then subjected to in vivo ubiquitination assay. S is short for starvation (f). Lysates of synchronized PC-3U cells treated with or without TRAF6 siRNA were subjected to immunoprecipitation using a Flag antibody, followed by immunoblotting using an HA antibody. Arrow points to heavy immunoglobulin chain. Data presented as mean±SEM, N=3 [Student's t-test, ** p < 0.01] (g) Lysates of synchronized PC-3U cells transfected with HA-tagged WT ubiquitin and Flag-tagged WT or mutant AURKB, were subjected to immunoprecipitation using a Flag antibody, followed by immunoblotting using an HA rabbit antibody. Data presented as mean±SEM, N=3 [Student's t-test, * p < 0.05]. (h) PC-3U cells were transfected with WT or mutant GFP-AURKB, and then subjected to immunoblotting with an antibody against H3pS10. Data presented as mean±SEM, N=3 [Student's t-test, ** p < 0.01]. (i) PC-3U cells were transfected with WT or mutant GFP-AURKB, then stained with TβRI (red). n=20, N=3, data presented as mean±SEM [Student's t-test, ** p < 0.01, *** p < 0.001]. (j) PC-3U cells were transfected with WT or mutant GFP-AURKB, then stained with Hoechst 33342. N=3 [Student's t-test, * p < 0.05]. (k) Schematic illustration of the kinase domain of AURKB. The structure file AF-Q96GD4-F1-model_V2pdb of human AURKB was downloaded from alphafold.ebi.ac.uk and uploaded to EzMol interface 2.1 (Imperial College London, UK) for visualization and depiction. The activation loop, the alpha C helixes (amino acid residues 110 to 131; alphaC´ (aa 110 to aa 115) and alphaC (aa 118 to 131)), K85 and K87, and G84, G86, G98, together with their corresponding side residues, are depicted in orange, magenta, red and blue, respectively. Note that the side chain of K85 and K87 (red) protrudes out from the kinase domain.

Article Snippet: Other primary antibodies against the following proteins were used in immunofluorescence experiments: AURKB (Novus, Cat# NBP2-50039, RRID:AB_2895237), and p-Smad2 (Cell Signaling Technology Cat# 3108, RRID:AB_490941).

Techniques: Blocking Assay, Western Blot, Incubation, Immunoprecipitation, Transfection, In Vivo, Ubiquitin Assay, Mutagenesis, Staining, Activation Assay

Journal: Oncogene

Article Title: Inhibition of Aurora A in response to DNA damage.

doi: 10.1038/sj.onc.1209056

Figure Lengend Snippet: Figure 4 DNA damage signal to AurA is relayed through Chk1. (a) Exponentially growing HeLa or HCT-15 cells were treated with etoposide (5 mM) for 4 h and Chk2 electrophoretic mobility (top) or kinase activity (bottom) was examined. (b) HCT-15 cells synchro- nized by double-thymidine block were treated in the presence of etoposide (5 mM) at 8 h postrelease and analysed at the indicated time points. The high level of Chk1-S345 phosphorylation at time 0 h likely reflected intra S phase checkpoint activation by stalled replication forks. (c) HeLa cells were treated with control or Chk1 siRNA and double-thymidine synchronized. Etoposide (5 mM) was given at 8 h and proteins analysed by Western blotting at 24 h postrelease. Expression of Chk1 was monitored using a rabbit polyclonal (top) or the DSC-310 mouse monoclonal antibody (bottom). (d) Synchronized HeLa cells were treated with the Chk1 inhibitor UCN-01 (300 nM) at 7.5 h postrelease. Etoposide (5 mM) was added at 8 h and cells were analysed at the indicated time points. Phosphorylation of CDK1 at Tyr15 was used as biochemical marker for the effect of UCN-01 (middle panel). The asterisk in AurA blot indicates a nonspecifically reacting protein that was taken as loading control. (e) Double-thymidine synchronized HeLa cells were treated with increasing amounts of UCN-01 (5–10–50– 100–300 nM, lanes 5–9) at time 7.5 h postrelease and DNA damage was generated by addition of etoposide (5 mM) at 8 h. The expression of AurA was examined at the indicated time points. The asterisk in AurA blot indicates a nonspecifically reacting protein that was taken as loading control.

Article Snippet: The purified polyclonal antibody to AurB (#3094) was from Cell Signaling Technology.

Techniques: Activity Assay, Blocking Assay, Phospho-proteomics, Activation Assay, Control, Western Blot, Expressing, Marker, Generated