Topoisomerase II alpha and beta (TOP2A and TOP2B) isoenzymes perform essential and non-redundant cellular functions. Anthracyclines induce their potent anti-cancer effects primarily via TOP2A, but at the same time they induce a dose limiting cardiotoxicity through TOP2B. Here we describe the development of the obex class of TOP2 inhibitors that bind to a previously unidentified druggable pocket in the TOP2 ATPase domain to act as allosteric catalytic inhibitors by locking the ATPase domain conformation with the capability of isoform-selective inhibition. Through rational drug design we have developed topobexin, which interacts with residues that differ between TOP2A and TOP2B to provide inhibition that is both selective for TOP2B and superior to dexrazoxane. Topobexin is a potent protectant against chronic anthracycline cardiotoxicity in an animal model. This demonstration of TOP2 isoform-specific inhibition underscores the broader potential to improve drug specificity and minimize adverse effects in various medical treatments.
© 2025. The Author(s).
Product Citations: 18
In Nature Communications on 28 May 2025 by Kubeš, J., Karabanovich, G., et al.
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In Cell Death & Disease on 23 January 2024 by Shu, J., Jiang, J., et al.
DNA topoisomerase II (TOP2) is an enzyme that performs a critical function in manipulating DNA topology during replication, transcription, and chromosomal compaction by forming a vital intermediate known as the TOP2-DNA cleavage complex (TOP2cc). Although the TOP2cc is often transient, stabilization can be achieved by TOP2 poisons, a family of anti-cancer chemotherapeutic agents targeting TOP2, such as etoposide (VP-16), and then induce double-strand breaks (DSBs) in cellular DNA. TOP2cc first needs to be proteolyzed before it can be processed by TDP2 for the removal of these protein adducts and to produce clean DNA ends necessary for proper repair. However, the mechanism by which TOP2βcc is proteolyzed has not been thoroughly studied. In this study, we report that after exposure to VP-16, MDM2, a RING-type E3 ubiquitin ligase, attaches to TOP2β and initiates polyubiquitination and proteasomal degradation. Mechanistically, during exposure to VP-16, TOP2β binds to DNA to form TOP2βcc, which promotes MDM2 binding and subsequent TOP2β ubiquitination and degradation, and results in a decrease in TOP2βcc levels. Biologically, MDM2 inactivation abrogates TOP2β degradation, stabilizes TOP2βcc, and subsequently increases the number of TOP2β-concealed DSBs, resulting in the rapid death of cancer cells via the apoptotic process. Furthermore, we demonstrate the combination activity of VP-16 and RG7112, an MDM2 inhibitor, in the xenograft tumor model and in situ lung cancer mouse model. Taken together, the results of our research reveal an underlying mechanism by which MDM2 promotes cancer cell survival in the presence of TOP2 poisons by activating proteolysis of TOP2βcc in a p53-independent manner, and provides a rationale for the combination of MDM2 inhibitors with TOP2 poisons for cancer therapy.
© 2024. The Author(s).
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Cell Biology
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RAD54L2 counters TOP2-DNA adducts to promote genome stability.
In Science Advances on 8 December 2023 by D'Alessandro, G., Morales-Juarez, D. A., et al.
The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.
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Genetics
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Topoisomerase II Inhibition Attenuates LPS-induced IL- 1β Secretion by Macrophages
Preprint on Research Square on 30 May 2023 by Brindle, A., Bainbridge, C., et al.
Inhibiting pathological secretion of Interleukin-1β has shown beneficial effects in disease models and in the clinic and thus there is interest in finding inhibitors that can reduce its release from macrophages in response to their activation by foreign pathogens. We used an in vitro human macrophage model to investigate whether ICRF-193, a Topoisomerase II inhibitor could modulate IL1B mRNA expression and IL-1β secretion. These macrophage-like cells readily secrete IL-1β in response to Lipopolysaccharide (LPS). Upon exposure to a non-toxic dose of ICRF-193, IL-1β secretion was diminished by ~ 40%; however, level of transcription of IL1B was unaffected. We show that there was no Topoisomerase 2B (TOP2B) binding to IL1B gene proximal sites, confirming that it is not involved directly in mediating the transcription of IL1B and hence why ICRF-193 does not alter IL1B mRNA levels. Quantification of Topoisomerase isoforms suggests that TOP2B plays a role in mediating the effects of ICRF-193 on IL-1β secretion. Hence, we show for the first time that ICRF-193 can reduce IL-1β secretion. Its low cost and the development of water-soluble prodrugs of ICRF-193 warrants its further investigation in the modulation of pathological secretion of this cytokine for the treatment of inflammatory disorders. (196 words)
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Replication-associated formation and repair of human topoisomerase IIIα cleavage complexes.
In Nature Communications on 6 April 2023 by Saha, L. K., Saha, S., et al.
Topoisomerase IIIα (TOP3A) belongs to the conserved Type IA family of DNA topoisomerases. Here we report that human TOP3A is associated with DNA replication forks and that a "self-trapping" TOP3A mutant (TOP3A-R364W) generates cellular TOP3A DNA cleavage complexes (TOP3Accs). We show that trapped TOP3Accs that interfere with replication, induce DNA damage and genome instability. To elucidate how TOP3Accs are repaired, we explored the role of Spartan (SPRTN), the metalloprotease associated with DNA replication, which digests proteins forming DNA-protein crosslinks (DPCs). We find that SPRTN-deficient cells show elevated TOP3Accs, whereas overexpression of SPRTN lowers cellular TOP3Accs. SPRTN is deubiquitinated and epistatic with TDP2 in response to TOP3Accs. In addition, we found that MRE11 can excise TOP3Accs, and that cell cycle determines the preference for the SPRTN-TDP2 vs. the ATM-MRE11 pathways, in S vs. G2, respectively. Our study highlights the prevalence of TOP3Accs repair mechanisms to ensure normal DNA replication.
© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.
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In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.1.C
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Homo sapiens (Human)
ATM signaling pathway is activated by VP-16 stimuli. (A, B) H1299 and A549 cells were incubated with various concentrations of VP-16 (0, 50, 100, and 250 μM) for 4 h, then, cells were collected, and subjected to immunoblotting (IB) with the indicated antibodies (Abs). ACTIN was used as a loading ...
more
ATM signaling pathway is activated by VP-16 stimuli. (A, B) H1299 and A549 cells were incubated with various concentrations of VP-16 (0, 50, 100, and 250 μM) for 4 h, then, cells were collected, and subjected to immunoblotting (IB) with the indicated antibodies (Abs). ACTIN was used as a loading control. (C, D) H1299 and A549 cells were treated with VP-16 (100 μM) for the indicated time periods (0, 1, 2, 4, and 8 h), followed by western blotting using the indicated Abs. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the decay curves are shown. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 1. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.1.B
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Homo sapiens (Human)
ATM signaling pathway is activated by VP-16 stimuli. (A, B) H1299 and A549 cells were incubated with various concentrations of VP-16 (0, 50, 100, and 250 μM) for 4 h, then, cells were collected, and subjected to immunoblotting (IB) with the indicated antibodies (Abs). ACTIN was used as a loading ...
more
ATM signaling pathway is activated by VP-16 stimuli. (A, B) H1299 and A549 cells were incubated with various concentrations of VP-16 (0, 50, 100, and 250 μM) for 4 h, then, cells were collected, and subjected to immunoblotting (IB) with the indicated antibodies (Abs). ACTIN was used as a loading control. (C, D) H1299 and A549 cells were treated with VP-16 (100 μM) for the indicated time periods (0, 1, 2, 4, and 8 h), followed by western blotting using the indicated Abs. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the decay curves are shown. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 1. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 30
In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.1.D
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Homo sapiens (Human)
ATM signaling pathway is activated by VP-16 stimuli. (A, B) H1299 and A549 cells were incubated with various concentrations of VP-16 (0, 50, 100, and 250 μM) for 4 h, then, cells were collected, and subjected to immunoblotting (IB) with the indicated antibodies (Abs). ACTIN was used as a loading ...
more
ATM signaling pathway is activated by VP-16 stimuli. (A, B) H1299 and A549 cells were incubated with various concentrations of VP-16 (0, 50, 100, and 250 μM) for 4 h, then, cells were collected, and subjected to immunoblotting (IB) with the indicated antibodies (Abs). ACTIN was used as a loading control. (C, D) H1299 and A549 cells were treated with VP-16 (100 μM) for the indicated time periods (0, 1, 2, 4, and 8 h), followed by western blotting using the indicated Abs. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the decay curves are shown. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 1. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.2.A
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Homo sapiens (Human)
KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretre...
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KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretreated with DMSO or KU60019 (5 μM) for 1 h, followed by treatment with VP-16 for various time periods, and then, IB was undertaken with the indicated Abs. (E, F) H1299 and A549 cells following treatment with CHX and VP-16 or with CHX, VP-16, and KU60019 (5 μM) for the respective times, cells were collected and IB with the relevant antibodies. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the decay curves are shown. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 2. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.2.C
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Homo sapiens (Human)
KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretre...
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KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretreated with DMSO or KU60019 (5 μM) for 1 h, followed by treatment with VP-16 for various time periods, and then, IB was undertaken with the indicated Abs. (E, F) H1299 and A549 cells following treatment with CHX and VP-16 or with CHX, VP-16, and KU60019 (5 μM) for the respective times, cells were collected and IB with the relevant antibodies. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the decay curves are shown. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 2. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 30
In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.2.B
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Homo sapiens (Human)
KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretre...
more
KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretreated with DMSO or KU60019 (5 μM) for 1 h, followed by treatment with VP-16 for various time periods, and then, IB was undertaken with the indicated Abs. (E, F) H1299 and A549 cells following treatment with CHX and VP-16 or with CHX, VP-16, and KU60019 (5 μM) for the respective times, cells were collected and IB with the relevant antibodies. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the decay curves are shown. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 2. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 30
In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.2.D
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Homo sapiens (Human)
KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretre...
more
KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretreated with DMSO or KU60019 (5 μM) for 1 h, followed by treatment with VP-16 for various time periods, and then, IB was undertaken with the indicated Abs. (E, F) H1299 and A549 cells following treatment with CHX and VP-16 or with CHX, VP-16, and KU60019 (5 μM) for the respective times, cells were collected and IB with the relevant antibodies. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the decay curves are shown. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 2. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 30
In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.2.F
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Homo sapiens (Human)
KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretre...
more
KU60019, an ATM inhibitor causes a dose and time-dependent accumulation of TOP2β. (A, B) H1299 and A549 cells were treated for 2 h with vehicle or VP-16 (100 μM) and varying doses of KU60019 (0, 1, 5, 10, and 20 μM) and then IB with the relevant antibodies. (C, D) H1299 and A549 cells were pretreated with DMSO or KU60019 (5 μM) for 1 h, followed by treatment with VP-16 for various time periods, and then, IB was undertaken with the indicated Abs. (E, F) H1299 and A549 cells following treatment with CHX and VP-16 or with CHX, VP-16, and KU60019 (5 μM) for the respective times, cells were collected and IB with the relevant antibodies. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the decay curves are shown. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 2. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 30
In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.4.H
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Homo sapiens (Human)
KU60019 increases the level of the TOP2β-DNA cleavage complex and impairs the repair of VP-16-induced DSBs. (A, B) H1299 cells were treated with VP-16 alone or in conjunction with KU60019 for the appropriate times, and then the TOP2cc levels were determined by FACS. (C, D) Two hours were spent tr...
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KU60019 increases the level of the TOP2β-DNA cleavage complex and impairs the repair of VP-16-induced DSBs. (A, B) H1299 cells were treated with VP-16 alone or in conjunction with KU60019 for the appropriate times, and then the TOP2cc levels were determined by FACS. (C, D) Two hours were spent treating H1299 cells with VP-16 or VP-16 in conjunction with KU60019 (5 μM). The cells were then collected for the TARDIS assay. (E, F) H1299 cells were pretreated with KU60019 (5 μM) for 1 h and then cotreated with VP-16 for an additional 1 h. Cells were then harvested for the neutral comet assay. Representative images are shown, and the data are presented as the mean ± S.D. from three independent experiments; ***p < 0.001. (G) Cells treated with VP-16 alone or in conjunction with KU60019 for the indicated time periods were harvested at the indicated time points for immunofluorescence. (H) H1299 and A549 cells were treated with KU60019 or VP-16 alone or in conjunction with IB with the indicated Abs. All experiments were independently repeated three times. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 4. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.6.C
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Homo sapiens (Human)
A combination of KU60019 and VP-16 promotes the apoptosis of lung cancer cells. (A, B) H1299 cells were treated with VP-16 (10 μM) alone or in conjunction with KU60019 (5 μM) for the indicated time periods and then subjected to FACS analysis to determine the apoptotic population. (C, D) H1299 and...
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A combination of KU60019 and VP-16 promotes the apoptosis of lung cancer cells. (A, B) H1299 cells were treated with VP-16 (10 μM) alone or in conjunction with KU60019 (5 μM) for the indicated time periods and then subjected to FACS analysis to determine the apoptotic population. (C, D) H1299 and A549 cells were incubated with various concentrations of KU60019 or VP-16 for 24 h, followed by MK-2206 (0.5 or 1 μM) or DMSO for 24 h. Cells were harvested for western blotting using indicated antibodies. All experiments were independently repeated three times. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 5. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.6.D
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Homo sapiens (Human)
A combination of KU60019 and VP-16 promotes the apoptosis of lung cancer cells. (A, B) H1299 cells were treated with VP-16 (10 μM) alone or in conjunction with KU60019 (5 μM) for the indicated time periods and then subjected to FACS analysis to determine the apoptotic population. (C, D) H1299 and...
more
A combination of KU60019 and VP-16 promotes the apoptosis of lung cancer cells. (A, B) H1299 cells were treated with VP-16 (10 μM) alone or in conjunction with KU60019 (5 μM) for the indicated time periods and then subjected to FACS analysis to determine the apoptotic population. (C, D) H1299 and A549 cells were incubated with various concentrations of KU60019 or VP-16 for 24 h, followed by MK-2206 (0.5 or 1 μM) or DMSO for 24 h. Cells were harvested for western blotting using indicated antibodies. All experiments were independently repeated three times. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 5. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
Image 1 of 30
In Sci Rep on 17 January 2023 by Shu, J., Wang, X., et al.
Fig.1.A
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Homo sapiens (Human)
ATM signaling pathway is activated by VP-16 stimuli. (A, B) H1299 and A549 cells were incubated with various concentrations of VP-16 (0, 50, 100, and 250 μM) for 4 h, then, cells were collected, and subjected to immunoblotting (IB) with the indicated antibodies (Abs). ACTIN was used as a loading ...
more
ATM signaling pathway is activated by VP-16 stimuli. (A, B) H1299 and A549 cells were incubated with various concentrations of VP-16 (0, 50, 100, and 250 μM) for 4 h, then, cells were collected, and subjected to immunoblotting (IB) with the indicated antibodies (Abs). ACTIN was used as a loading control. (C, D) H1299 and A549 cells were treated with VP-16 (100 μM) for the indicated time periods (0, 1, 2, 4, and 8 h), followed by western blotting using the indicated Abs. All experiments were independently repeated three times. Densitometry quantification was performed with Image J, and the decay curves are shown. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 1. Molecular weight markers are noted to the left of blot figure.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Oncotarget on 19 February 2022 by Menendez, D., Anand, J. R., et al.
Fig.1.A
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p53 functional status influences etoposide - induced Top2-DNA complexes.(A) The ICE assay was performed in isogenic HCT116 cell lines following etoposide treatment for 1 hr at the doses indicated; representative immunoblots for Top2α, Top2β and dsDNA are shown. (B) Quantification of Top2cc per ce...
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p53 functional status influences etoposide - induced Top2-DNA complexes.(A) The ICE assay was performed in isogenic HCT116 cell lines following etoposide treatment for 1 hr at the doses indicated; representative immunoblots for Top2α, Top2β and dsDNA are shown. (B) Quantification of Top2cc per cell. P values <0.05 were considered significant (*).
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Collected and cropped from Oncotarget by CiteAb, provided under a CC-BY license
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In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.1.A
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TOP2β is a novel substrate of CRL E3 ligases.a MLN4924 treatment blocks VM-26-induced TOP2β degradation. Cells were left untreated or treated with VM-26 (100 μM) or in combination with MLN4924 (1 μM) for 2 h. Cells were then harvested, and immunoblotting (IB) was undertaken using the indicated an...
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TOP2β is a novel substrate of CRL E3 ligases.a MLN4924 treatment blocks VM-26-induced TOP2β degradation. Cells were left untreated or treated with VM-26 (100 μM) or in combination with MLN4924 (1 μM) for 2 h. Cells were then harvested, and immunoblotting (IB) was undertaken using the indicated antibodies (Abs). b MLN4924 increases TOP2β levels in a dose-dependent manner. Cells were treated with various concentrations of MLN4924 for 24 h, and then, IB was undertaken with the indicated Abs. c MLN4924 treatment extends the protein half-life of TOP2β upon VM-26 treatment. Cells were treated with CHX (100 μg/ml) and VM-26 (100 μM) or in combination with MLN4924 (1 μM) for the indicated time periods, and then, IB was undertaken with the indicated Abs (top). Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom). d MLN4924 treatment suppresses the polyubiquitination of TOP2β induced by VM-26 treatment. HEK293 cells transfected with the indicated plasmids were treated with VM-26 (100 μM) and MG132 (20 μM) or in combination with MLN4924 (1 μM) for 5 h, and then, IP was conducted with anti-HA beads (top), and direct IB was undertaken with the indicated Abs (bottom). WCE: whole-cell extract. e RBX1 binds to endogenous TOP2β. HEK293 cells transfected with the indicated plasmids were harvested for IP with anti-FLAG beads (top) and direct IB with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
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In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.1.E
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TOP2β is a novel substrate of CRL E3 ligases.a MLN4924 treatment blocks VM-26-induced TOP2β degradation. Cells were left untreated or treated with VM-26 (100 μM) or in combination with MLN4924 (1 μM) for 2 h. Cells were then harvested, and immunoblotting (IB) was undertaken using the indicated an...
more
TOP2β is a novel substrate of CRL E3 ligases.a MLN4924 treatment blocks VM-26-induced TOP2β degradation. Cells were left untreated or treated with VM-26 (100 μM) or in combination with MLN4924 (1 μM) for 2 h. Cells were then harvested, and immunoblotting (IB) was undertaken using the indicated antibodies (Abs). b MLN4924 increases TOP2β levels in a dose-dependent manner. Cells were treated with various concentrations of MLN4924 for 24 h, and then, IB was undertaken with the indicated Abs. c MLN4924 treatment extends the protein half-life of TOP2β upon VM-26 treatment. Cells were treated with CHX (100 μg/ml) and VM-26 (100 μM) or in combination with MLN4924 (1 μM) for the indicated time periods, and then, IB was undertaken with the indicated Abs (top). Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom). d MLN4924 treatment suppresses the polyubiquitination of TOP2β induced by VM-26 treatment. HEK293 cells transfected with the indicated plasmids were treated with VM-26 (100 μM) and MG132 (20 μM) or in combination with MLN4924 (1 μM) for 5 h, and then, IP was conducted with anti-HA beads (top), and direct IB was undertaken with the indicated Abs (bottom). WCE: whole-cell extract. e RBX1 binds to endogenous TOP2β. HEK293 cells transfected with the indicated plasmids were harvested for IP with anti-FLAG beads (top) and direct IB with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.1.B
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TOP2β is a novel substrate of CRL E3 ligases.a MLN4924 treatment blocks VM-26-induced TOP2β degradation. Cells were left untreated or treated with VM-26 (100 μM) or in combination with MLN4924 (1 μM) for 2 h. Cells were then harvested, and immunoblotting (IB) was undertaken using the indicated an...
more
TOP2β is a novel substrate of CRL E3 ligases.a MLN4924 treatment blocks VM-26-induced TOP2β degradation. Cells were left untreated or treated with VM-26 (100 μM) or in combination with MLN4924 (1 μM) for 2 h. Cells were then harvested, and immunoblotting (IB) was undertaken using the indicated antibodies (Abs). b MLN4924 increases TOP2β levels in a dose-dependent manner. Cells were treated with various concentrations of MLN4924 for 24 h, and then, IB was undertaken with the indicated Abs. c MLN4924 treatment extends the protein half-life of TOP2β upon VM-26 treatment. Cells were treated with CHX (100 μg/ml) and VM-26 (100 μM) or in combination with MLN4924 (1 μM) for the indicated time periods, and then, IB was undertaken with the indicated Abs (top). Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom). d MLN4924 treatment suppresses the polyubiquitination of TOP2β induced by VM-26 treatment. HEK293 cells transfected with the indicated plasmids were treated with VM-26 (100 μM) and MG132 (20 μM) or in combination with MLN4924 (1 μM) for 5 h, and then, IP was conducted with anti-HA beads (top), and direct IB was undertaken with the indicated Abs (bottom). WCE: whole-cell extract. e RBX1 binds to endogenous TOP2β. HEK293 cells transfected with the indicated plasmids were harvested for IP with anti-FLAG beads (top) and direct IB with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.1.C
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WB
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Homo sapiens (Human)
TOP2β is a novel substrate of CRL E3 ligases.a MLN4924 treatment blocks VM-26-induced TOP2β degradation. Cells were left untreated or treated with VM-26 (100 μM) or in combination with MLN4924 (1 μM) for 2 h. Cells were then harvested, and immunoblotting (IB) was undertaken using the indicated an...
more
TOP2β is a novel substrate of CRL E3 ligases.a MLN4924 treatment blocks VM-26-induced TOP2β degradation. Cells were left untreated or treated with VM-26 (100 μM) or in combination with MLN4924 (1 μM) for 2 h. Cells were then harvested, and immunoblotting (IB) was undertaken using the indicated antibodies (Abs). b MLN4924 increases TOP2β levels in a dose-dependent manner. Cells were treated with various concentrations of MLN4924 for 24 h, and then, IB was undertaken with the indicated Abs. c MLN4924 treatment extends the protein half-life of TOP2β upon VM-26 treatment. Cells were treated with CHX (100 μg/ml) and VM-26 (100 μM) or in combination with MLN4924 (1 μM) for the indicated time periods, and then, IB was undertaken with the indicated Abs (top). Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom). d MLN4924 treatment suppresses the polyubiquitination of TOP2β induced by VM-26 treatment. HEK293 cells transfected with the indicated plasmids were treated with VM-26 (100 μM) and MG132 (20 μM) or in combination with MLN4924 (1 μM) for 5 h, and then, IP was conducted with anti-HA beads (top), and direct IB was undertaken with the indicated Abs (bottom). WCE: whole-cell extract. e RBX1 binds to endogenous TOP2β. HEK293 cells transfected with the indicated plasmids were harvested for IP with anti-FLAG beads (top) and direct IB with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.2.E
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WB
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Homo sapiens (Human)
β-TrCP binds to TOP2β and negatively regulates TOP2β levels.a Evolutionary conservation of the β-TrCP degron motifs of TOP2β. b β-TrCP silencing abrogates TOP2β reduction by VM-26 treatment. Cells transfected with siRNA oligos targeting both β-TrCP1 and β-TrCP2 (siβ-TrCP1 + 2) or scrambled contro...
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β-TrCP binds to TOP2β and negatively regulates TOP2β levels.a Evolutionary conservation of the β-TrCP degron motifs of TOP2β. b β-TrCP silencing abrogates TOP2β reduction by VM-26 treatment. Cells transfected with siRNA oligos targeting both β-TrCP1 and β-TrCP2 (siβ-TrCP1 + 2) or scrambled control siRNA (siCtrl) were left untreated or treated with VM-26 (100 μM) for 2 h, and then, IB was undertaken with the indicated Abs. c β-TrCP silencing suppresses the TOP2β degradation by VM-26 treatment in a time-dependent manner but has no effect on TOP2α. Cells transfected with siRNA oligos targeting β-TrCP or scrambled control siRNA were treated with VM-26 for the indicated time periods, and then, IB was undertaken with the indicated Abs. d β-TrCP silencing extends TOP2β protein half-life upon VM-26 treatment. Cells transfected with the indicated siRNA were treated with CHX and VM-26 for the indicated time periods and then subjected to IB with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom). e Ectopically expressed β-TrCP1 binds to endogenous TOP2β. HEK293 cells transfected with the indicated plasmids were left untreated or treated with VM-26 and MG132 (20 μM) for 5 h. Cells were then harvested for IP with anti-FLAG beads (top) and direct IB with the indicated Abs (bottom). f β-TrCP1 binds to endogenous TOP2β. HeLa cells were lysed and subjected to IP with β-TrCP1 antibody or normal rabbit immunoglobulin (IgG), and then, IB was undertaken with anti-TOP2β Ab (top). Cell lysate was also directly immunoblotted with the indicated Abs (bottom). g β-TrCP1 promotes TOP2β polyubiquitination in vivo. HEK293 cells transfected with the indicated plasmids were treated with MG132 and VM-26 for 5 h, and then, IP was conducted with anti-HA beads (top), and direct IB was undertaken with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.2.C
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WB
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Homo sapiens (Human)
β-TrCP binds to TOP2β and negatively regulates TOP2β levels.a Evolutionary conservation of the β-TrCP degron motifs of TOP2β. b β-TrCP silencing abrogates TOP2β reduction by VM-26 treatment. Cells transfected with siRNA oligos targeting both β-TrCP1 and β-TrCP2 (siβ-TrCP1 + 2) or scrambled contro...
more
β-TrCP binds to TOP2β and negatively regulates TOP2β levels.a Evolutionary conservation of the β-TrCP degron motifs of TOP2β. b β-TrCP silencing abrogates TOP2β reduction by VM-26 treatment. Cells transfected with siRNA oligos targeting both β-TrCP1 and β-TrCP2 (siβ-TrCP1 + 2) or scrambled control siRNA (siCtrl) were left untreated or treated with VM-26 (100 μM) for 2 h, and then, IB was undertaken with the indicated Abs. c β-TrCP silencing suppresses the TOP2β degradation by VM-26 treatment in a time-dependent manner but has no effect on TOP2α. Cells transfected with siRNA oligos targeting β-TrCP or scrambled control siRNA were treated with VM-26 for the indicated time periods, and then, IB was undertaken with the indicated Abs. d β-TrCP silencing extends TOP2β protein half-life upon VM-26 treatment. Cells transfected with the indicated siRNA were treated with CHX and VM-26 for the indicated time periods and then subjected to IB with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom). e Ectopically expressed β-TrCP1 binds to endogenous TOP2β. HEK293 cells transfected with the indicated plasmids were left untreated or treated with VM-26 and MG132 (20 μM) for 5 h. Cells were then harvested for IP with anti-FLAG beads (top) and direct IB with the indicated Abs (bottom). f β-TrCP1 binds to endogenous TOP2β. HeLa cells were lysed and subjected to IP with β-TrCP1 antibody or normal rabbit immunoglobulin (IgG), and then, IB was undertaken with anti-TOP2β Ab (top). Cell lysate was also directly immunoblotted with the indicated Abs (bottom). g β-TrCP1 promotes TOP2β polyubiquitination in vivo. HEK293 cells transfected with the indicated plasmids were treated with MG132 and VM-26 for 5 h, and then, IP was conducted with anti-HA beads (top), and direct IB was undertaken with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.2.F
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WB
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Homo sapiens (Human)
β-TrCP binds to TOP2β and negatively regulates TOP2β levels.a Evolutionary conservation of the β-TrCP degron motifs of TOP2β. b β-TrCP silencing abrogates TOP2β reduction by VM-26 treatment. Cells transfected with siRNA oligos targeting both β-TrCP1 and β-TrCP2 (siβ-TrCP1 + 2) or scrambled contro...
more
β-TrCP binds to TOP2β and negatively regulates TOP2β levels.a Evolutionary conservation of the β-TrCP degron motifs of TOP2β. b β-TrCP silencing abrogates TOP2β reduction by VM-26 treatment. Cells transfected with siRNA oligos targeting both β-TrCP1 and β-TrCP2 (siβ-TrCP1 + 2) or scrambled control siRNA (siCtrl) were left untreated or treated with VM-26 (100 μM) for 2 h, and then, IB was undertaken with the indicated Abs. c β-TrCP silencing suppresses the TOP2β degradation by VM-26 treatment in a time-dependent manner but has no effect on TOP2α. Cells transfected with siRNA oligos targeting β-TrCP or scrambled control siRNA were treated with VM-26 for the indicated time periods, and then, IB was undertaken with the indicated Abs. d β-TrCP silencing extends TOP2β protein half-life upon VM-26 treatment. Cells transfected with the indicated siRNA were treated with CHX and VM-26 for the indicated time periods and then subjected to IB with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom). e Ectopically expressed β-TrCP1 binds to endogenous TOP2β. HEK293 cells transfected with the indicated plasmids were left untreated or treated with VM-26 and MG132 (20 μM) for 5 h. Cells were then harvested for IP with anti-FLAG beads (top) and direct IB with the indicated Abs (bottom). f β-TrCP1 binds to endogenous TOP2β. HeLa cells were lysed and subjected to IP with β-TrCP1 antibody or normal rabbit immunoglobulin (IgG), and then, IB was undertaken with anti-TOP2β Ab (top). Cell lysate was also directly immunoblotted with the indicated Abs (bottom). g β-TrCP1 promotes TOP2β polyubiquitination in vivo. HEK293 cells transfected with the indicated plasmids were treated with MG132 and VM-26 for 5 h, and then, IP was conducted with anti-HA beads (top), and direct IB was undertaken with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.4.B
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WB
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Homo sapiens (Human)
ATM binds with and phosphorylates TOP2β at Ser1134 to promote its degradation by VM-26.a Inhibition of ATM, but not ATR or DNA-PK, blocks VM-26-induced TOP2β degradation. Cells were pretreated with KU60019, AZD6738, or LTURM34 for 1 h and then treated with VM-26 for an additional 2 h. Cells were ...
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ATM binds with and phosphorylates TOP2β at Ser1134 to promote its degradation by VM-26.a Inhibition of ATM, but not ATR or DNA-PK, blocks VM-26-induced TOP2β degradation. Cells were pretreated with KU60019, AZD6738, or LTURM34 for 1 h and then treated with VM-26 for an additional 2 h. Cells were then harvested for IB with the indicated Abs. b–d Inhibition of ATM extends the protein half-life of TOP2β. SK-BR3 and MDA-MB231 cells were pretreated with DMSO or KU60019 (5 μM) for 1 h (b) or transfected with the indicated siRNA (c), followed by treatment with CHX and VM-26. Atm WT or KO MEFs (d) were also treated with CHX and VM-26 for various time periods, and then, IB was undertaken with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom, b; right, c and d). e TOP2β binds to endogenous ATM. HEK293 cells were transfected with the indicated plasmids, and then, IP was conducted with anti-FLAG beads (top), and direct IB was undertaken with the indicated Abs (bottom). f Evolutionary conservation of two putative ATM phosphorylation sites (in red) on TOP2β is shown. g ATM phosphorylates TOP2β at Ser1134. HEK293 cells transfected with the indicated plasmids were left untreated or treated with VM-26, AZD6738, and LTURM34 for 5 h. Cells were harvested after MG132 treatment for 5 h, and then, IP was conducted using anti-FLAG Abs (top), and direct IB was undertaken with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.4.C
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WB
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Homo sapiens (Human)
ATM binds with and phosphorylates TOP2β at Ser1134 to promote its degradation by VM-26.a Inhibition of ATM, but not ATR or DNA-PK, blocks VM-26-induced TOP2β degradation. Cells were pretreated with KU60019, AZD6738, or LTURM34 for 1 h and then treated with VM-26 for an additional 2 h. Cells were ...
more
ATM binds with and phosphorylates TOP2β at Ser1134 to promote its degradation by VM-26.a Inhibition of ATM, but not ATR or DNA-PK, blocks VM-26-induced TOP2β degradation. Cells were pretreated with KU60019, AZD6738, or LTURM34 for 1 h and then treated with VM-26 for an additional 2 h. Cells were then harvested for IB with the indicated Abs. b–d Inhibition of ATM extends the protein half-life of TOP2β. SK-BR3 and MDA-MB231 cells were pretreated with DMSO or KU60019 (5 μM) for 1 h (b) or transfected with the indicated siRNA (c), followed by treatment with CHX and VM-26. Atm WT or KO MEFs (d) were also treated with CHX and VM-26 for various time periods, and then, IB was undertaken with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom, b; right, c and d). e TOP2β binds to endogenous ATM. HEK293 cells were transfected with the indicated plasmids, and then, IP was conducted with anti-FLAG beads (top), and direct IB was undertaken with the indicated Abs (bottom). f Evolutionary conservation of two putative ATM phosphorylation sites (in red) on TOP2β is shown. g ATM phosphorylates TOP2β at Ser1134. HEK293 cells transfected with the indicated plasmids were left untreated or treated with VM-26, AZD6738, and LTURM34 for 5 h. Cells were harvested after MG132 treatment for 5 h, and then, IP was conducted using anti-FLAG Abs (top), and direct IB was undertaken with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.5.E
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WB
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Homo sapiens (Human)
CK1 binds with and phosphorylates TOP2β at Ser1130 to promote its degradation by VM-26.a Inhibition of CK1, but not CK2 or GSK3, blocks the degradation of TOP2β induced by VM-26. Cells pretreated with D4476, CX-4945 or GSK3i-IX for 1 h were treated with VM-26 for an additional 2 h. Cells were the...
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CK1 binds with and phosphorylates TOP2β at Ser1130 to promote its degradation by VM-26.a Inhibition of CK1, but not CK2 or GSK3, blocks the degradation of TOP2β induced by VM-26. Cells pretreated with D4476, CX-4945 or GSK3i-IX for 1 h were treated with VM-26 for an additional 2 h. Cells were then harvested for IB analysis with the indicated Abs. b Inhibition of CK1 extends the protein half-life of TOP2β. Cells left untreated or pretreated with D4476 (50 μM) for 4 h were then exposed to CHX and VM-26. Cells were harvested at the indicated time points for IB analysis with the indicated Abs (left). Densitometry quantification was performed with ImageJ, and the decay curves are shown (right). c and d Silencing of CK1 inhibits TOP2β degradation by extending its protein half-life. Cells transfected with the indicated siRNA oligos were treated with VM-26 for 1 h (c) or treated with VM-26 and CHX for the indicated time periods (d), and then, IB was undertaken with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (d, right). e, f CK1 binds to and phosphorylates TOP2β at Ser1130. HEK293 cells transfected with the indicated plasmids were treated with VM-26 for 5 h as indicated (e) or in combination with MG132 (f), and then, IP was conducted with anti-FLAG beads (top), and direct IB was undertaken with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.5.C
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WB
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Homo sapiens (Human)
CK1 binds with and phosphorylates TOP2β at Ser1130 to promote its degradation by VM-26.a Inhibition of CK1, but not CK2 or GSK3, blocks the degradation of TOP2β induced by VM-26. Cells pretreated with D4476, CX-4945 or GSK3i-IX for 1 h were treated with VM-26 for an additional 2 h. Cells were the...
more
CK1 binds with and phosphorylates TOP2β at Ser1130 to promote its degradation by VM-26.a Inhibition of CK1, but not CK2 or GSK3, blocks the degradation of TOP2β induced by VM-26. Cells pretreated with D4476, CX-4945 or GSK3i-IX for 1 h were treated with VM-26 for an additional 2 h. Cells were then harvested for IB analysis with the indicated Abs. b Inhibition of CK1 extends the protein half-life of TOP2β. Cells left untreated or pretreated with D4476 (50 μM) for 4 h were then exposed to CHX and VM-26. Cells were harvested at the indicated time points for IB analysis with the indicated Abs (left). Densitometry quantification was performed with ImageJ, and the decay curves are shown (right). c and d Silencing of CK1 inhibits TOP2β degradation by extending its protein half-life. Cells transfected with the indicated siRNA oligos were treated with VM-26 for 1 h (c) or treated with VM-26 and CHX for the indicated time periods (d), and then, IB was undertaken with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (d, right). e, f CK1 binds to and phosphorylates TOP2β at Ser1130. HEK293 cells transfected with the indicated plasmids were treated with VM-26 for 5 h as indicated (e) or in combination with MG132 (f), and then, IP was conducted with anti-FLAG beads (top), and direct IB was undertaken with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30
In Oncogenesis on 3 February 2020 by Shu, J., Cui, D., et al.
Fig.5.D
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WB
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Homo sapiens (Human)
CK1 binds with and phosphorylates TOP2β at Ser1130 to promote its degradation by VM-26.a Inhibition of CK1, but not CK2 or GSK3, blocks the degradation of TOP2β induced by VM-26. Cells pretreated with D4476, CX-4945 or GSK3i-IX for 1 h were treated with VM-26 for an additional 2 h. Cells were the...
more
CK1 binds with and phosphorylates TOP2β at Ser1130 to promote its degradation by VM-26.a Inhibition of CK1, but not CK2 or GSK3, blocks the degradation of TOP2β induced by VM-26. Cells pretreated with D4476, CX-4945 or GSK3i-IX for 1 h were treated with VM-26 for an additional 2 h. Cells were then harvested for IB analysis with the indicated Abs. b Inhibition of CK1 extends the protein half-life of TOP2β. Cells left untreated or pretreated with D4476 (50 μM) for 4 h were then exposed to CHX and VM-26. Cells were harvested at the indicated time points for IB analysis with the indicated Abs (left). Densitometry quantification was performed with ImageJ, and the decay curves are shown (right). c and d Silencing of CK1 inhibits TOP2β degradation by extending its protein half-life. Cells transfected with the indicated siRNA oligos were treated with VM-26 for 1 h (c) or treated with VM-26 and CHX for the indicated time periods (d), and then, IB was undertaken with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (d, right). e, f CK1 binds to and phosphorylates TOP2β at Ser1130. HEK293 cells transfected with the indicated plasmids were treated with VM-26 for 5 h as indicated (e) or in combination with MG132 (f), and then, IP was conducted with anti-FLAG beads (top), and direct IB was undertaken with the indicated Abs (bottom).
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Collected and cropped from Oncogenesis by CiteAb, provided under a CC-BY license
Image 1 of 30