Measurement of Cdk1/Cyclin B Kinase Activity by Specific Antibodies and Western Blotting

Lewis, Cody W.; Taylor, Ryan G.; Golsteyn, Roy M.

doi:10.1007/978-1-4939-2957-3_21

Cited by 6 publications

(2 citation statements)

References 10 publications

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“…Cdk1 kinase assays were completed as outlined in Lewis and colleagues (42,43). Briefly, 20 ng of glutathione-S-transferase (GST) fused with a 9 amino acid PP1Ca peptide (GRPITPPRN) was combined with 2,000 cells in 2x Cdk1 phospho-buffer (100 mmol/L b-glycerophosphate, 20 mmol/L MgCl 2 , 20 mmol/L NaF, and 2 mmol/L DTT) and 400 mmol/L ATP and then incubated at 37 C for 15 minutes.…”

Section: Kinase Assaymentioning

confidence: 99%

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition

et al. 2019

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Adavosertib (also known as AZD1775 or MK1775) is a small-molecule inhibitor of the protein kinase Wee1, with single-agent activity in multiple solid tumors, including sarcoma, glioblastoma, and head and neck cancer. Adavosertib also shows promising results in combination with genotoxic agents such as ionizing radiation or chemotherapy. Previous studies have investigated molecular mechanisms of primary resistance to Wee1 inhibition. Here, we investigated mechanisms of acquired resistance to Wee1 inhibition, focusing on the role of the Wee1-related kinase Myt1. Myt1 and Wee1 kinases were both capable of phosphorylating and inhibiting Cdk1/ cyclin B, the key enzymatic complex required for mitosis, demonstrating their functional redundancy. Ectopic activa-tion of Cdk1 induced aberrant mitosis and cell death by mitotic catastrophe. Cancer cells with intrinsic adavosertib resistance had higher levels of Myt1 compared with sensitive cells. Furthermore, cancer cells that acquired resistance following short-term adavosertib treatment had higher levels of Myt1 compared with mock-treated cells. Downregulating Myt1 enhanced ectopic Cdk1 activity and restored sensitivity to adavosertib. These data demonstrate that upregulating Myt1 is a mechanism by which cancer cells acquire resistance to adavosertib.

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Section: Kinase Assaymentioning

confidence: 99%

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition

et al. 2019

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“…Nonspecific sites in the membrane are then blocked with BSA, non-fat milk powder, or casein. Finally, a labeled secondary antibody is added for detection by chemiluminescence or fluorescence (Lewis et al, 2016). WBA has been used for the confirmation of presence of purified proteins produced against various pathological antigens such as the lethal toxin of Clostridium sordellii (Arya et al, 2013), shiga toxin Stx2f (Skinner et al, 2013), Staphylococcus aureus alpha-hemolysin (alphatoxin) (Ladhani et al, 2001), Selenocosmia huwena huwentoxin-IV (HWTX-IV) , and V. parahaemolyticus thermolabile hemolysin (TLH) .…”

Section: Western Blotmentioning

confidence: 99%

Antibody Engineering for Pursuing a Healthier Future

et al. 2017

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Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.

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Experimental Determination of Checkpoint Adaptation by Mitotic Shake-Off and Microscopy

Swift

Golsteyn

2018

Methods in Molecular Biology

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Cells that undergo checkpoint adaptation arrest at and then abrogate the G2/M cell cycle checkpoint to enter mitosis with damaged DNA. Cells surviving this process frequently contain micronuclei, which can lead to genomic change and chromothripsis. In this chapter we describe how to induce checkpoint adaptation and detect it by time-lapse video and immunofluorescence microscopy and how to isolate cells undergoing checkpoint adaptation from a total cell population.

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Measurement of Cdk1/Cyclin B Kinase Activity by Specific Antibodies and Western Blotting

Cited by 6 publications

References 10 publications

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition

Antibody Engineering for Pursuing a Healthier Future

Experimental Determination of Checkpoint Adaptation by Mitotic Shake-Off and Microscopy

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