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  1. Article: Antibody-free detection of cellular neddylation dynamics of Cullin1

    Schwinn, Marie K / Jingya Ma / Keith V. Wood / Michael E. Bembenek / Ping Li / Trish Hoang / William D. Mallender / Xiansi Zhao / Xiaofeng Yang / Zhong-Hua Yan

    Analytical biochemistry. 2018 Aug. 15, v. 555

    2018  

    Abstract: Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay ... ...

    Abstract Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time.
    Keywords cell lines ; constitutive photomorphogenesis 9 signalosome ; post-translational modification ; viability
    Language English
    Dates of publication 2018-0815
    Size p. 67-72.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 1110-1
    ISSN 1096-0309 ; 0003-2697
    ISSN (online) 1096-0309
    ISSN 0003-2697
    DOI 10.1016/j.ab.2018.05.002
    Database NAL-Catalogue (AGRICOLA)

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    In stock of ZB MED Bonn / Germany

    Z 70/124: Show issues

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  2. Article: An ultrasensitive assay format for detecting ULK1 inhibition by monitoring the phosphorylation status of Atg13

    Yan, Zhong-Hua / Dongyun Wu / Jie Yu / Jiejin Chen / Johara Chouitar / Michael E. Bembenek / Neil Rollins / Olga Tayber / Pam Brauer / Robin Bosse / Wenhai Zhang

    Analytical biochemistry. 2016 Sept. 15, v. 509

    2016  

    Abstract: A new technology from Quanterix called SiMoA (single molecule array) which employs a fully automated system capable of ultrasensitive sandwich based ELISA detection was explored. Our studies focused upon the inhibition of the autophagy initiating kinase ... ...

    Abstract A new technology from Quanterix called SiMoA (single molecule array) which employs a fully automated system capable of ultrasensitive sandwich based ELISA detection was explored. Our studies focused upon the inhibition of the autophagy initiating kinase ULK1 by measuring the both total Atg13 and the phosphorylation of Atg13(pSer318) from control and following compound treatment in either overexpressing or wild type tissue culture samples. The results show linear protein concentration dependence over two orders of magnitude and provide an assay window of 8- to 100-fold signal to background for inhibition of phosphorylation for both wild type and overexpressed samples, respectively. Moreover, overexpressed samples displayed 17-fold pSer318-Atg13 above wild type levels of with no apparent differences in compound potency. Lastly, the inhibition of ULK1 from mouse derived wild type xenografts also demonstrated loss of pSer318-Atg13 upon ULK1 inhibitor treatment that compared favorably to Western blot. These results show that the SiMoA technology can detect quantitatively low levels of endogenous biomarkers with the ability to detect the loss of pSer318-Atg13 upon ULK1 inhibition.
    Keywords autophagy ; biomarkers ; enzyme-linked immunosorbent assay ; mice ; monitoring ; phosphorylation ; technology ; tissue culture ; Western blotting
    Language English
    Dates of publication 2016-0915
    Size p. 73-78.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 1110-1
    ISSN 1096-0309 ; 0003-2697
    ISSN (online) 1096-0309
    ISSN 0003-2697
    DOI 10.1016/j.ab.2016.06.023
    Database NAL-Catalogue (AGRICOLA)

    More links

    Kategorien

    In stock of ZB MED Bonn / Germany

    Z 70/124: Show issues

    Order via subito

    This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.

    Inter-library loan at ZB MED

    Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.

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