recombinant human aurkb  (SignalChem)

Journal: Nature Communications

Article Title: Aurora kinase B phosphorylates ZBP1 to drive PANoptosis following treatment with PARP and ATR inhibitors combination

doi: 10.1038/s41467-025-66591-1

Figure Lengend Snippet: A Immunoprecipitation assay indicates ZBP1 phosphorylation levels in 293T cells transfected with Myc-tagged ZBP1 and HA-tagged AURKB or its phosphonull TA mutants. Immunoprecipitated ZBP1 was probed with anti-phosphor-ser/thr antibody. B A schematic of the human ZBP1 sequence highlighting the phosphorylation sites observed by MS is shown. Fold changes in the abundance of phosphorylated peptides of ZBP1 in 293T cells transfected with AURKB-WT relative to 293T cells transfected with vectors are shown. Dots indicate data from three independent experiments. Additionally, fold changes in the abundance of phosphorylated peptides of ZBP1 in 293T cells transfected with AURKB-TA relative to 293T cells transfected with AURKB-WT are shown. C , D After the KD of ZBP1, OVCAR8 cells were transfected with plasmids expressing Myc-tagged ZBP1 or its phosphonull mutants and then were subjected to 250 nM Talazoparib and 2.5 μM Ceralasertib for 24 h. Immunoblot analysis of ZBP1, GSDME, PARP1, pMLKL, and tMLKL. GAPDH was used as the internal control. E The S309 phosphorylation site of ZBP1 in 293T cells after treated with 250 nM Talazoparib and 2.5 μM Ceralasertib for 24 h was analyzed by LC-MS/MS. F – I After the KD of ZBP1, OVCAR8 cells were transfected with plasmids expressing Myc-tagged ZBP1 or its phosphonull S309A mutants and then were subjected to 250 nM Talazoparib and 2.5 μM Ceralasertib for 24 h. F Phase-contrast imaging assay of OVCAR8 cells. The red arrowheads indicate the large bubbles emerging from the plasma membrane. Scale bar = 100 μm. Representative images ( G ) and quantification ( H ) of the PI+ cells (red) and YP1+ cells (green). Scale bar = 100 μm. I Comparison of LDH release-based cell death. J The validation of ZBP1-S309 antibody using dot-blot assay. The vertical axis represents the concentration of the antibody. K In vitro phosphorylation assay of ZBP1 by purified protein AURKB or CDK1. L Immunoblot analysis in OVCAR8 cells after treated by 250 nM Talazoparib and 2.5 μM Ceralasertib and/or 1 μM Barasertib for 24 h. M After treating with 250 nM Talazoparib and 2.5 μM Ceralasertib for 24 h, OVCAR8 cells were shaken off to enrich mitotic cells and then treated with 1 μM Barasertib for 6 h. Immunoblot analysis of pAURKB, AURKB, pZBP1, and ZBP1 in OVCAR8 cells. GAPDH was used as the internal control. Representative images ( N ) and quantification ( O ) of the PLA to detect the physical association between AURKB and pZBP1-S309. Scale bar = 20 μm. P CO-IP assay to assess the interactions among ZBP1, RIPK1, CASP8, and CASP6 after transfected with Myc-tagged ZBP1 or its phosphonull S309A mutants and treated with 250 nM Talazoparib and 2.5 μM Ceralasertib in 293T cells. CASP8: caspase 8, CASP6: caspase 6. Q Schematic overview of the mechanism of DDR inhibitors-induced PANoptosis during aberrant mitotic progression. Created in BioRender. Liao, Z. (2025) https://BioRender.com/bstbek5 . The data in ( H , I , O ) are representative of three independent experiments and presented as the mean ± SEM. Data were repeated thrice independently in ( A , C , D , F , K – M , P ) with similar results. Statistical significance was determined using two-tailed Student’s t test in ( O ) and one-way ANOVA with Tukey’s multiple comparisons test in ( H , I ). Source data are provided as a file.

Article Snippet: Flag-tagged recombinant human AURKB protein was purchased from Proteintech (Cat: 81352), GST-tagged recombinant human CDK1 & Cyclin B1 protein was obtained from SinoBiological (Cat: C22B-10G), and GST-tagged recombinant human ZBP1 protein was obtained from CUSABI (Cat: CSB- EP861990 ).

Techniques: Immunoprecipitation, Phospho-proteomics, Transfection, Sequencing, Expressing, Western Blot, Control, Liquid Chromatography with Mass Spectroscopy, Imaging, Clinical Proteomics, Membrane, Comparison, Biomarker Discovery, Dot Blot, Concentration Assay, In Vitro, Purification, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: Cancer discovery

Article Title: AKT degradation selectively inhibits the growth of PI3K/PTEN pathway mutant cancers with wild type KRAS and BRAF by destabilizing Aurora kinase B

doi: 10.1158/2159-8290.CD-20-0815

Figure Lengend Snippet: A. IB analysis of indicated protein levels in PC-3 cells were treated with DMSO or indicated compounds at 1 μM for 24 hr. B-C. (B) Cell cycle analysis by flow cytometry of PC-3 cells after 48 hr treatment with DMSO, AZD5363, MS21, or GDC-0941. Histograms are representative of triplicates. (C) Quantification of cell cycle phases of PC-3 cells after 48 hr treatment as indicated. Percentages reflect the mean of triplicates, and error bars indicate SEM. D. Indicated cells were treated with DMSO or MS21 at 0.1 μM, 0.3 μM, 1 μM, 3 μM, or 10 μM for 2 weeks. Cells were stained with Crystal Violet. E. In vitro AKT/PKB phosphorylation of AURKB. Recombinant active full-length human AKT1/PKB protein (400 ng) and recombinant active full-length human AURKB protein (500 ng) were incubated with or without 200 μM ATP in 50 μL 1 × kinase buffer at 30°C for 30 min. 600 μM of Barasertib or AZD5363 were pre-incubated with recombinant protein for 20 min before ATP was added into the reactions. IB analysis of phosphorylation of AURKB using anti-Phospho-AKT Substrate (RXXS*/T*) antibody after kinase assay. F. IP of HA-AURKB protein and IB analysis of phospho-AKT substrate in 293T-AURKB-T232A or 293T-AURKB-T73A/T232A cells treated with Insulin (200 μg/mL) for 0, 10, 30, 60 mins after starvation for overnight. G. IP of HA-AURKB and IB analysis of phosphor-AKT substrate in 293T-HA-AURKB cells pretreated with or without GDC0941 (1 μM) for 10 mins and followed by insulin (10 μg/mL) stimulation for 10 mins. H. IP of HA-AURKB and IB analysis of phospho-AKT substrate in PC-3-AURKB cells treated with DMSO or AZD5363 (1 μM) for 2 hr. I. IP of endogenous AURKB and IB analysis of phospho-AKT substrate in PC-3 cells pretreated with or without AZD5363 (1 μM) for 20 mins and followed by insulin (200 μg/mL) stimulation for 10 mins. J. Indicated cells were treated with DMSO or MS21 at 3 μM for 24 hr and lysates tested for AURKB, T-AKT. K. IB analysis of AURKB protein in PC-3 cells treated with GDC0941 (1 μM) and MG132 (10 μM) for indicated times.

Article Snippet: AKT kinase assay: Recombinant active full-length human Akt1/PKB protein (400 ng) (Cat#14–276, Millipore Sigma) was incubated with 200 μM ATP (Cat#20–306, Sigma) and 500 ng recombinant active full-length human AURKB protein (Cat#14–835, Millipore Sigma) in 50 μl 1 × kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2, Cat#9802, Cell Signaling Technology).

Techniques: Cell Cycle Assay, Flow Cytometry, Staining, In Vitro, Phospho-proteomics, Recombinant, Incubation, Kinase Assay