Journal: Genome biology

Article Title: Functional screening reveals genetic dependencies and diverging cell cycle control in atypical teratoid rhabdoid tumors.

doi: 10.1186/s13059-024-03438-w

Figure Lengend Snippet: Fig. 7 AMBRA1 regulates ubiquitin-dependent degradation of aurora kinases in a context-dependent manner. A Complex structure prediction using AlphaFold2_mmseqs2 for AMBRA1WD40 with DDB1, cyclin D1, AURKA or AURKB. Top, cartoon illustration of predicted heteromeric protein complexes. Interchain AlphaFold2 contacts (< 8 Å) are shown as straight lines colored by predicted alignment error (PAE). AMBRA1 residues implicated in DDB1 binding are highlighted in green. Predicted template modeling scores (pTM) are indicated. Bottom, pairwise PAE scores for all protein complexes. B Heat map showing log2 fold changes of label-free quantification values from FLAG affinity purification and mass spectrometry detection in AMBRA-FLAG versus FLAG expressing ATRT cells. Cells were investigated with and without treatment of the CRL inhibitor MLN4924. C Co-immunoprecipitation analyses followed by western blot for selected potential AMBRA1 interactors and substrates. Previously identified interactors and substrates of AMBRA1 (CUL4A, DDB1, and cyclin D1) were included as controls. D Top: Immunoassays of cyclin D1, cyclin D3, AURKA, and AURKB for BT12 and CHLA06 cells, both in wildtype and AMBRA1-knockout conditions. Cells were treated with DMSO, 0.4 μM Baf-A1, or 1 μM MLN4924 for 4 h (cyclin D) or 12 h (aurora kinases). Increase in LC3B-II levels was used as a validation of autophagy inhibition. Bottom: Quantification of protein expression levels relative the corresponding DMSO control conditions. E Left: Immunoassays from His pull-down experiments for BT12 and CHLA266 cells, both in wildtype and AMBRA1-knockout conditions, transfected either with 6xHis-empty or 6xHis-tagged ubiquitin 48 h prior to pull-down. Right: Quantification of AURKA and AURKB ubiquitylation relative to total protein levels. F Model of context-dependent, CRL4AMBRA1-associated blockade of cell cycle regulators via degradation by the ubiquitin-proteasome system. Data are shown as mean ± SD (D, E). Statistics are derived from two-way ANOVA tests with Dunnett’s (D) or Sidak’s correction (E). *P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001

Article Snippet: Primary antibodies used in this study for DigiWest were the following: β-actin (Sigma, A1978, AMBRA1 (Cell Signaling, 24907), ATM (Cell Signaling, 2873), ATR (Cell Signaling, 2790), AURKA (Cell Signaling, 4718), AURKB (Cell Signaling, 3094), BUB1B (Cell Signaling, 5421), Caspase3 (Cell Signaling, 9662), CDK1 (Cell Signaling, 9112), CDK1-pTyr15 (Cell Signaling, 4539), CDC25A (abm, Y021163), CDC25A-pSer75 (abm, Y011138), CDC25B (R&D, AF1649), CDC27 (Transduction Laboratories, C40920), CDK2 (Cell Signaling, 2546), CDK2-pThr160 (Cell Signaling, 2561), CDK3 (abcam, ab135805), CDK4 (Cell Signaling, 2906), CDK4-pThr172 (Invitrogen, PA-64482), CDK5 (Cell Signaling, 2506), CDK6 (Cell Signaling, 13331), CDK6-pTyr13 (biorbyt, orb15013), CDK6-pTyr24 (biorbyt, orb15014), CHK2 (Cell Signaling, 3440), CHK2-pThr68 (Cell Signaling, 2661), c-MYC (Cell Signaling, 9402), c-MYC-pThr58 (ThermoFisher, PA5-37654), c-MYC-pThr62/Ser62 (abcam, ab32029), cyclin A (abcam, ab53054), cyclin B1 (abcam, ab32053), cyclin D1 (Cell Signaling, 2926), cyclin D1-pThr286 (ThermoFisher, PA5-37487), cyclin D2 (Cell Signaling, 3741), cyclin D3 (Cell Signaling, 2936), cyclin E1 (Cell Signaling, 4129), cyclin E2 (Cell Signaling, 4132), E2F-2 (Millipore, DR1095), E2F-4 (biorbyt, orb10571), histone H3-pSer28 (Millipore, 07–145), hisotne H3-pSer10 (Cell Signaling, 9701), MCM2 (Cell Signaling, 3619), MCM2-pSer139 (Cell Signaling, 8861), MDM2 (Santa Cruz, sc-965), MDM2-pSer166 (Cell Signaling, 3521), p16 (ProteinTech Group, 10883-1-AP), p21 (Cell Signaling, 2947), p27 (Cell Signaling, 3698), p53 (Santa Cruz, sc-126), p53-pSer37 (Cell Signaling, 9289), p53-pSer15 (Cell Signaling, 9284), RB (Cell Signaling, 9313), RB-pSer807/Ser811 (Cell Signaling, 8516), RB-pSer780 (Cell Signaling, 3590), RB-pSer608 (Cell Signaling, 8147), RB-pSer795 (Cell Signaling, 9301), RBPSUH (Cell Signaling, 5313), RPA2 p34 (Millipore, 04–1481), Survivin (Cell Signaling, 2802), TOPK (Cell Signaling, 4942), and TOPO 2 alpha (Santa Cruz, sc-13058).

Techniques: Ubiquitin Proteomics, Binding Assay, Quantitative Proteomics, Affinity Purification, Mass Spectrometry, Expressing, Immunoprecipitation, Western Blot, Knock-Out, Biomarker Discovery, Inhibition, Control, Transfection, Derivative Assay

Journal: Cell reports

Article Title: A High-Content Screen Identifies TPP1 and Aurora B as Regulators of Axonal Mitochondrial Transport

doi: 10.1016/j.celrep.2019.08.035

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pCMV6-Myc-DDK-Aurkb (human) , OriGene , RC210288.

Techniques: Recombinant, Software

Journal: PLoS ONE

Article Title: Generation of Human Induced Pluripotent Stem Cells Using Epigenetic Regulators Reveals a Germ Cell-Like Identity in Partially Reprogrammed Colonies

doi: 10.1371/journal.pone.0082838

Figure Lengend Snippet: Human blastocysts were incubated with primary antibodies for ( A ) DNMT3B (green), AURKB (orange) and SETD7 (red) or ( B ) PRMT5 (green), AURKB (orange) and SETD7 (red), the nuclear DNA stained with DAPI (blue) and visualized by multi-channel confocal microscopy. Differential Interference Contrast (DIC) was used to distinguish the inner cell mass (ICM) from the trophoectoderm of each embryo. Note that the expression of DNMT3B, AURKB and PRMT5 primarily localized to the ICM of human blastocysts, whereas SETD7 expression was predominantly detected in the outer trophoectoderm layer of the embryos (indicated by white arrows).

Article Snippet: While the AURKB goat polyclonal antibody (catalog #AF4006) was purchased from R&D Systems, Inc. (Minneapolis, MN), the PRMT5 rabbit monoclonal antibody (clone #EPR5772) was obtained from Epitomics (Burlingame, CA).

Techniques: Incubation, Staining, Confocal Microscopy, Expressing

Journal: PLoS ONE

Article Title: Generation of Human Induced Pluripotent Stem Cells Using Epigenetic Regulators Reveals a Germ Cell-Like Identity in Partially Reprogrammed Colonies

doi: 10.1371/journal.pone.0082838

Figure Lengend Snippet: The expression of Histone H3-S28P (green), H4-R3me2 (red; middle panel) and H3-K4me3 (red; right panel), which are primarily mediated by AURKB, PRMT5 and SETD7, respectively, was evaluated in ( A ) undifferentiated and ( B ) Day 7 differentiated hiPSCs (HUF5 clone 2) by immunofluorescent confocal microscopy. Note that H3-K4me3 expression is increased or at least retained in hiPSCs upon differentiation. ( C ) Similar confocal analysis of histone modifications in Day 7 differentiated hiPSCs demonstrating that only a small population of cells still expresses H3-S28P, to suggest that they are proliferative, and decreased expression of H4-R3me2 in hiPSCs following 7 days of differentiation.

Article Snippet: While the AURKB goat polyclonal antibody (catalog #AF4006) was purchased from R&D Systems, Inc. (Minneapolis, MN), the PRMT5 rabbit monoclonal antibody (clone #EPR5772) was obtained from Epitomics (Burlingame, CA).

Techniques: Expressing, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: List of reagents and antibodies.

Article Snippet: 8 , Anti-AURKB antibody , 1:500 , NBP261493 , Novus Biologicals.

Techniques: Concentration Assay, SYBR Green Assay, Protease Inhibitor, Saline, Plasmid Preparation, Magnetic Beads, Membrane