Quantification of Dicer Activity in Mammalian Cell Lysates Using a Non-radioactive Fluorescence Method

2016 ◽  
pp. 21-27
Author(s):  
Anna Emde ◽  
Natalia Rivkin ◽  
Mark A. Behlke ◽  
Eran Hornstein
Keyword(s):  
Mammalian Cell ◽  
Fluorescence Method ◽  
Cell Lysates

scholarly journals Direct Interaction and Reciprocal Regulation between ASK1 and Calcineurin-NFAT Control Cardiomyocyte Death and Growth

2006 ◽  
Vol 26 (10) ◽  
pp. 3785-3797 ◽  
Author(s):  
Qinghang Liu ◽  
Benjamin J. Wilkins ◽  
Yong J. Lee ◽  
Hidenori Ichijo ◽  
Jeffery D. Molkentin
Keyword(s):  
Mammalian Cell ◽  
Calcium Binding ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Adaptive Responses ◽  
Reciprocal Regulation ◽  
Hypertrophic Growth ◽  
Cell Lysates ◽  
Calcineurin B ◽  
Induced Apoptosis

ABSTRACT The calcium-calmodulin-activated protein phosphatase calcineurin functions as a key mediator of diverse biologic processes, including differentiation, apoptosis, growth, and adaptive responses, in part through dephosphorylation and activation of nuclear factor of activated T-cell (NFAT) transcription factors. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream component of the mitogen-activated protein kinases that serves as a pivotal regulator of cytokine-, oxidative-, and stress-induced cell death. Here, we performed a yeast two-hybrid screen with calcineurin B as bait, which identified ASK1 as a direct physical interacting partner. The C-terminal 218 amino acids of ASK1 were sufficient to mediate interaction with calcineurin B in yeast, as well as in mammalian cell lysates. Importantly, endogenous calcium binding B subunit (CnB) protein interacted with endogenous ASK1 protein in cardiomyocytes at baseline, suggesting that the interaction observed in yeast was of potential biologic relevance. Indeed, calcineurin directly dephosphorylated ASK1 at serine 967 using purified proteins or mammalian cell lysates. Dephosphorylation of ASK1 serine 967 by calcineurin promoted its disassociation from 14-3-3 proteins, resulting in ASK1 activation. Calcineurin and ASK1 cooperatively enhanced cardiomyocyte apoptosis, while expression of a dominant negative ASK1 blocked calcineurin-induced apoptosis. Mouse embryonic fibroblasts deficient in ask1 were also partially resistant to calcineurin- or ionomycin-induced apoptosis. Finally, ASK1 negatively regulated calcineurin-NFAT signaling indirectly through c-Jun NH2-terminal kinase (JNK)- and p38-mediated phosphorylation of NFAT, which blocked calcineurin- and agonist-dependent hypertrophic growth of cardiomyocytes. Thus, ASK1 and calcineurin-NFAT constitute a feedback regulatory circuit in which calcineurin positively regulates ASK1 through direct dephosphorylation, while ASK1 negatively regulates calcineurin-NFAT signaling through p38- and JNK-mediated NFAT phosphorylation.

Analysis of Single Mammalian Cell Lysates by Mass Spectrometry

1996 ◽  
Vol 118 (46) ◽  
pp. 11662-11663 ◽  
Author(s):  
Liang Li ◽  
Rafael E. Golding ◽  
Randy M. Whittal
Keyword(s):  
Mass Spectrometry ◽  
Mammalian Cell ◽  
Cell Lysates

Comparative proteomic profiling of mammalian cell lysates using phosphopeptide microarrays

2012 ◽  
Vol 48 (16) ◽  
pp. 2240 ◽  
Author(s):  
Liqian Gao ◽  
Mahesh Uttamchandani ◽  
Shao Q. Yao
Keyword(s):  
Mammalian Cell ◽  
Proteomic Profiling ◽  
Cell Lysates

Rapid and reliable preparation of total cell lysates from fission yeast

2006 ◽  
Author(s):  
Atsuko Shirai ◽  
Akihisa Matsuyama ◽  
Yoko Yashiroda ◽  
Ritsuko Arai ◽  
Minoru Yoshida
Keyword(s):  
Fission Yeast ◽  
Total Cell ◽  
Cell Lysates
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