A western blot assay to measure cyclin dependent kinase activity in cells or in vitro without the use of radioisotopes

Lewis, Cody W.; Taylor, Ryan G.; Kubara, Philip M.; Marshall, Kris; Meijer, Laurent; Golsteyn, Roy M.

doi:10.1016/j.febslet.2013.08.003

Cited by 35 publications

(27 citation statements)

References 19 publications

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“…2). Cdk1 activity was estimated by monitoring the phosphorylation at residue T320 of a peptide derived from the protein phosphatase 1 subunit PPP1CC (GST-PP1C-S), as described in Lewis et al (2013) (Fig. 2).…”

Section: Cdc4mentioning

confidence: 99%

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Fine-tuning of Cdc6 accumulation by Cdk1 and MAP kinase is essential for completion of oocyte meiotic divisions

Daldello

Poulhe

et al. 2015

Journal of Cell Science

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).In Fig. 4B, the western blot used to illustrate the elution profile of Cdk1 in the control was mistakenly used a second time to illustrate the samples incubated with anti-Cdc6 antibody. The correct Fig. 4 is shown below. This error does not affect the conclusions of the study.The authors apologise to readers for any confusion that this error might have caused. , Olivier Haccard 1,2 and Aude Dupré1 ,2, * ABSTRACT Vertebrate oocytes proceed through the first and the second meiotic division without an intervening S-phase to become haploid. Although DNA replication does not take place, unfertilized oocytes acquire the competence to replicate DNA one hour after the first meiotic division by accumulating an essential factor of the replicative machinery, Cdc6. Here, we show that the turnover of Cdc6 is precisely regulated in oocytes to avoid inhibition of Cdk1. At meiosis resumption, Cdc6 is expressed but cannot accumulate owing to a degradation mechanism that is activated through Cdk1. During transition from the first to the second meiotic division, Cdc6 is under the antagonistic regulation of B-type cyclins (which interact with and stabilize Cdc6) and the Mos-MAPK pathway (which negatively controls Cdc6 accumulation). Because overexpressing Cdc6 inhibits Cdk1 reactivation and drives oocytes into a replicative interphasic state, the fine-tuning of Cdc6 accumulation is essential to ensure two meiotic waves of Cdk1 activation and to avoid unscheduled DNA replication during meiotic maturation.

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Section: Cdc4mentioning

confidence: 99%

“…GSTp21 Cip1 and GST-PP1C-S were produced by using autoinduction (Studier, 2005) and purified as previously described (Frank-Vaillant et al, 1999;Lewis et al, 2013).…”

Section: Expression and Purification Of Gst-p21mentioning

confidence: 99%

Fine-tuning of Cdc6 accumulation by Cdk1 and MAP kinase is essential for completion of oocyte meiotic divisions

Daldello

Poulhe

et al. 2015

Journal of Cell Science

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“…49 M059K or WI-38 cells were cultivated at 4,000 cells per well on a 96 well plate for 48 hours prior to treatment. Results were expressed as IC 50 , which was the compound concentration that reduced the absorbance by 50% at 590 nm, compared to DMSO treated cells. All measurements were done in triplicate.…”

Section: Cytotoxicity Assaysmentioning

confidence: 99%

“…Western blots were performed at least 2 times. 50 Immunofluorescence microscopy Cells were plated on glass coverslips for 48 h prior to treatment, then fixed in 3% formaldehyde for 20 min at room temperature. Fixation was quenched by addition of 50 mM NH 4 Cl in PBS, the cells were permeabilized for 5 min in 0.2% Triton X-100, and blocked with 3% BSA for 30 min.…”

Section: Electrophoresis and Protein Gel Blottingmentioning

confidence: 99%

Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA

Lewis

Golsteyn

2016

Cell Cycle

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We have examined the relationship between checkpoint adaptation (mitosis with damaged DNA) and micronuclei. Micronuclei in cancer cells are linked to genomic change, and may induce chromothripsis (chromosome shattering). We measured the cytotoxicity of the cancer drug cisplatin in M059K (glioma fibroblasts, IC50 15 mM). Nearly 100% of M059K cells were positive for histone gH2AX staining after 48 h treatment with a cytotoxic concentration of cisplatin. The proportion of micronucleated cells, as confirmed by microscopy using DAPI and lamin A/C staining, increased from 24% to 48%, and the total micronuclei in surviving cells accumulated over time. Promoting entry into mitosis with a checkpoint inhibitor increased the number of micronuclei in cells whereas blocking checkpoint adaptation with a Cdk inhibitor reduced the number of micronuclei. Interestingly, some micronuclei underwent asynchronous DNA replication, relative to the main nuclei, as measured by deoxy-bromo-uracil (BrdU) staining. These micronuclei stained positive for histone gH2AX, which was linked to DNA replication, suggesting that micronuclei arise from checkpoint adaptation and that micronuclei may continue to damage DNA. By contrast the normal cell line WI-38 did not undergo checkpoint adaptation when treated with cisplatin and did not show changes in micronuclei number. These data reveal that the production of micronuclei by checkpoint adaptation is part of a process that contributes to genomic change.

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“…Western blot assay was performed according to the methods of Lewis et al (22) and VallejoIllarramendi et al (23). Briefly, A549 cells were seeded into 48-well plates.…”

Section: Myeloperoxidase (Mpo) Assaysmentioning

confidence: 99%

Anti-inflammatory effects of triptolide by inhibiting the NF-κB signalling pathway in LPS-induced acute lung injury in a murine model

Xian

Zhang

Duan

et al. 2014

Molecular Medicine Reports

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Abstract.Triptolide is one of the main active components in the Chinese herb Tripterygium wilfordii Hook F, which has been demonstrated to possess anti-inflammatory properties. The aim of this study was to investigate the effects of triptolide on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and to explore the possible mechanisms. Mice were administered LPS intranasally to induce lung injury, and triptolide was administered intraperitoneally 1 h prior to the LPS challenge. Triptolide-treated mice exhibited significantly reduced levels of leukocytes, myeloperoxidase activity and edema of the lung, as well as tumour necrosis factor-α, interleukin (IL)-1β and IL-6 production in the bronchoalveolar lavage fluid compared with LPS-treated mice. Additionally, western blot analysis showed that triptolide inhibited the LPS-induced phosphorylation of nuclear factor of κ light polypeptide gene enhancer in B cells inhibitor-α and nuclear factor κ-light-chain-enhancer of activated B cells-p65 (NF-κB p65) and the expression of Toll-like receptor 4 (TLR4). In conclusion, the results from the present study suggest that the anti-inflammatory effect of triptolide against LPS-induced ALI may be due to its ability to inhibit the TLR4-mediated NF-κB signalling pathway. Triptolide may therefore be a promising potential therapeutic agent for ALI treatment, which may ultimately aid the clinical therapy for patients with ALI.

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A western blot assay to measure cyclin dependent kinase activity in cells or in vitro without the use of radioisotopes

Cited by 35 publications

References 19 publications

Fine-tuning of Cdc6 accumulation by Cdk1 and MAP kinase is essential for completion of oocyte meiotic divisions

Fine-tuning of Cdc6 accumulation by Cdk1 and MAP kinase is essential for completion of oocyte meiotic divisions

Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA

Anti-inflammatory effects of triptolide by inhibiting the NF-κB signalling pathway in LPS-induced acute lung injury in a murine model

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