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.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 Mol+Cell+Proteomics
2015 ; 14
(3
): 707-23
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Monitoring interactions and dynamics of endogenous beta-catenin with
intracellular nanobodies in living cells
#MMPMID25595278
Traenkle B
; Emele F
; Anton R
; Poetz O
; Haeussler RS
; Maier J
; Kaiser PD
; Scholz AM
; Nueske S
; Buchfellner A
; Romer T
; Rothbauer U
Mol Cell Proteomics
2015[Mar]; 14
(3
): 707-23
PMID25595278
show ga
?-catenin is the key component of the canonical Wnt pathway and plays a crucial
role in a multitude of developmental and homeostatic processes. The different
tasks of ?-catenin are orchestrated by its subcellular localization and
participation in multiprotein complexes. To gain a better understanding of
?-catenin's role in living cells we have generated a new set of single domain
antibodies, referred to as nanobodies, derived from heavy chain antibodies of
camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal
domain of ?-catenin and applied these new high-affinity binders as capture
molecules in sandwich immunoassays and co-immunoprecipitations of endogenous
?-catenin complexes. In addition, we engineered intracellularly functional
anti-?-catenin chromobodies by combining the binding moieties of the nanobodies
with fluorescent proteins. For the first time, we were able to visualize the
subcellular localization and nuclear translocation of endogenous ?-catenin in
living cells using these chromobodies. Moreover, the chromobody signal allowed us
to trace the accumulation of diffusible, hypo-phosphorylated ?-catenin in
response to compound treatment in real time using High Content Imaging. The
anti-?-catenin nanobodies and chromobodies characterized in this study are
versatile tools that enable a novel and unique approach to monitor the dynamics
of subcellular ?-catenin in biochemical and cell biological assays.
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