Analysis of In Vitro DNA Interactions of Brassinosteroid-Controlled Transcription Factors Using Electrophoretic Mobility Shift Assay

2017 ◽  
pp. 133-144 ◽  
Author(s):  
Simon J. Unterholzner ◽  
Wilfried Rozhon ◽  
Brigitte Poppenberger
Keyword(s):  
Transcription Factors ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Dna Interactions ◽  
Mobility Shift ◽  
Mobility Shift Assay

scholarly journals Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin β promoter

2008 ◽  
Vol 42 (3) ◽  
pp. 225-237 ◽  
Author(s):  
Yumiko Kashiwabara ◽  
Shigekazu Sasaki ◽  
Akio Matsushita ◽  
Koji Nagayama ◽  
Kenji Ohba ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Minimal Sequence ◽  
Mobility Shift Assay ◽  
Β Chain

Thyrotropin (TSH) is a heterodimer consisting of α and β chains, and the β chain (TSHβ) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHβ expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHβ promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHβ promoter without PIT1. The deleted region (nt −82/−52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHβ promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

scholarly journals A detailed and radioisotope-free protocol for electrophoretic mobility shift assay (EMSA)

2021 ◽  
Author(s):  
NGUYEN HOAI NGUYEN
Keyword(s):  
Transcription Factor ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Chromatin Immunoprecipitation ◽  
Direct Interaction ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Dna Fragment ◽  
Mobility Shift Assay

Abstract To comprehensively characterize the functions of a transcription factor (TF), it is required to analyze the interaction of this TF with its targeted loci. Several methods such as β-glucuronidase (GUS) or luciferase reporter, yeast one-hybrid (Y1H), chromatin-immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) assays have been developed. Of these, EMSA is an in vitro method which can prove the direct interaction between TF and targeted DNA fragment. This protocol is to provide a detailed procedure for a safe EMSA assay (without using any radioisotope).

scholarly journals From an electrophoretic mobility shift assay to isolated transcription factors: a fast genomic-proteomic approach

BMC Genomics ◽  
2010 ◽  
Vol 11 (1) ◽  
Author(s):  
Astrid R Mach-Aigner ◽  
Karin Grosstessner-Hain ◽  
Marcio J Poças-Fonseca ◽  
Karl Mechtler ◽  
Robert L Mach
Keyword(s):  
Transcription Factors ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Mobility Shift ◽  
Proteomic Approach ◽  
Mobility Shift Assay

A fluorescence based non-radioactive electrophoretic mobility shift assay

2000 ◽  
Vol 78 (2) ◽  
pp. 163-170 ◽  
Author(s):  
K Ruscher ◽  
M Reuter ◽  
D Kupper ◽  
G Trendelenburg ◽  
U Dirnagl ◽  
...  
Keyword(s):  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Mobility Shift ◽  
Mobility Shift Assay
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