scholarly journals Acyl-PEGyl exchange gel-shift (APEGS) assay for palmitoylation quantification

2021 ◽  
Vol 65 (2) ◽  
pp. 41-45
Author(s):  
Masaki Fukata ◽  
Norihiko Yokoi ◽  
Yuko Fukata
Keyword(s):  
Gel Shift

A General EMSA (Gel-shift) Protocol

BIO-PROTOCOL ◽  
2011 ◽  
Vol 1 (3) ◽  
Author(s):  
Ran Chen
Keyword(s):  
Gel Shift

scholarly journals Phosphorylation of MgrA and Its Effect on Expression of the NorA and NorB Efflux Pumps of Staphylococcus aureus

2010 ◽  
Vol 192 (10) ◽  
pp. 2525-2534 ◽  
Author(s):  
Que Chi Truong-Bolduc ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Multidrug Resistance ◽  
Virulence Factors ◽  
Double Mutant ◽  
Efflux Pumps ◽  
Efflux Transporters ◽  
Global Regulator ◽  
Binding Assays ◽  
Genes Encoding ◽  
Gel Shift

ABSTRACT MgrA is a global regulator in Staphylococcus aureus that controls the expression of diverse genes encoding virulence factors and multidrug resistance (MDR) efflux transporters. We identified pknB, which encodes the (Ser/Thr) kinase PknB, in the S. aureus genome. PknB was able to autophosphorylate as well as phosphorylate purified MgrA. We demonstrated that rsbU, which encodes a Ser/Thr phosphatase and is involved in the activation of the SigB regulon, was able to dephosphorylate MgrA-P but not PknB-P. Serines 110 and 113 of MgrA were found to be phosphorylated, and Ala substitutions at these positions resulted in reductions in the level of phosphorylation of MgrA. DNA gel shift binding assays using norA and norB promoters showed that MgrA-P was able to bind the norB promoter but not the norA promoter, a pattern which was the reverse of that for unphosphorylated MgrA. The double mutant MgrAS110A-S113A bound to the norA promoter but not the norB promoter. The double mutant led to a 2-fold decrease in norA transcripts and a 2-fold decrease in the MICs of norfloxacin and ciprofloxacin in strain RN6390. Thus, phosphorylation of MgrA results in loss of binding to the norA promoter, but with a gain of the ability to bind the norB promoter. Loss of the ability to phosphorylate MgrA by Ala substitution resulted in increased repression of norA expression and in reductions in susceptibilities to NorA substrates.

scholarly journals Application of Cu(I)-catalyzed azide–alkyne cycloaddition for the design and synthesis of sequence specific probes targeting double-stranded DNA

2016 ◽  
Vol 12 ◽  
pp. 1348-1360 ◽  
Author(s):  
Svetlana V Vasilyeva ◽  
Vyacheslav V Filichev ◽  
Alexandre S Boutorine
Keyword(s):  
Minor Groove ◽  
Binding Parameters ◽  
Design And Synthesis ◽  
Minor Groove Binders ◽  
Double Stranded Dna ◽  
Triple Helices ◽  
Gel Shift ◽  
Groove Binders

Efficient protocols based on Cu(I)-catalyzed azide–alkyne cycloaddition were developed for the synthesis of conjugates of pyrrole–imidazole polyamide minor groove binders (MGB) with fluorophores and with triplex-forming oligonucleotides (TFOs). Diverse bifunctional linkers were synthesized and used for the insertion of terminal azides or alkynes into TFOs and MGBs. The formation of stable triple helices by TFO-MGB conjugates was evaluated by gel-shift experiments. The presence of MGB in these conjugates did not affect the binding parameters (affinity and triplex stability) of the parent TFOs.

scholarly journals Gel Shift and UV Cross-linking Analysis ofTetrahymenaTelomerase

1995 ◽  
Vol 270 (15) ◽  
pp. 8893-8901 ◽  
Author(s):  
Lea Harrington ◽  
Christina Hull ◽  
Jill Crittenden ◽  
Carol Greider
Keyword(s):  
Cross Linking ◽  
Gel Shift

Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation of (alpha)5(beta)1 integrin expression

2000 ◽  
Vol 113 (18) ◽  
pp. 3173-3185 ◽  
Author(s):  
M.K. Duncan ◽  
Z. Kozmik ◽  
K. Cveklova ◽  
J. Piatigorsky ◽  
A. Cvekl
Keyword(s):  
Transgenic Mice ◽  
Binding Sites ◽  
Fiber Cell ◽  
Regulatory Sequences ◽  
Pax6 Gene ◽  
Fiber Cells ◽  
Nuclear Extracts ◽  
Shift Analysis ◽  
Beta 1 Integrin ◽  
Gel Shift

The PAX6 gene, a key regulator of eye development, produces two major proteins that differ in paired domain structure: PAX6 and PAX6(5a). It is known that an increase in the PAX6(5a) to PAX6 ratio leads to multiple ocular defects in humans. Here, transgenic mice were created that overexpress human PAX6(5a) in the lens. These mice develop cataracts with abnormalities in fiber cell shape as well as fiber cell/lens capsule and fiber cell/fiber cell interactions. While the structure of the actin cytoskeleton appeared relatively normal, the cataractous lens expresses increased amounts of paxillin and p120(ctn) as well as large aggregates of (alpha)5(beta)1 integrin in the dysgenic fiber cells. The elevated amounts of these proteins in the transgenic lens correlated well with elevated levels of their respective mRNAs. To investigate the role of Pax-6(5a) in the upregulation of these genes, a series of gel shift experiments using truncated proteins and consensus oligonucleotides demonstrated the complexity of Pax-6 and Pax-6(5a) binding to DNA, aiding our identification of potential binding sites in the human (α)5- and (beta)1-integrin promoters. Consequent gel shift analysis demonstrated that these putative regulatory sequences bind Pax-6 and/or Pax-6(5a) in lens nuclear extracts, suggesting that the human (alpha)5 and (beta)1 integrin promoters contain PAX6/PAX6(5a) binding sites and maybe directly regulated by this transcription factor in the transgenic lens. We conclude that these transgenic mice are good models to study a type of human cataract and for identifying batteries of genes that are directly or indirectly regulated by both forms of Pax-6.

scholarly journals LUEGO: a cost and time saving gel shift procedure

BioTechniques ◽  
2011 ◽  
Vol 51 (4) ◽  
pp. 267-269 ◽  
Author(s):  
Nicolas Jullien ◽  
Jean-Paul Herman
Keyword(s):  
Time Saving ◽  
Gel Shift

scholarly journals Analysis of transcription factors by gel shift and in-gel kinase assays-Gel shift assay.

1998 ◽  
Vol 42 (2) ◽  
pp. 93-96
Author(s):  
Takumi Kawabe ◽  
Toshifumi Tetsuka ◽  
Takashi Okamoto
Keyword(s):  
Transcription Factors ◽  
Gel Shift Assay ◽  
Gel Shift

scholarly journals Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Yong Feng ◽  
Yunfei Liao ◽  
Jianming Zhang ◽  
Jacson Shen ◽  
Zengwu Shao ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Transcriptional Analysis ◽  
Luciferase Assay ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Gel Shift Assay ◽  
Gel Shift

Abstract Background Aberrant expression of cyclin-dependent protein kinases (CDK) is a hallmark of cancer. CDK11 plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 and CDK11 transcriptionally regulated genes are largely unknown. Methods In this study, we performed a global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional targets of CDK11. Clinical relevance and function of core-binding factor subunit beta (CBFβ) were further accessed in osteosarcoma. Results We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of CBFβ expression in osteosarcoma cells. The CBFβ promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFβ is a direct transcriptional target of CDK11. High expression of CBFβ is associated with poor outcome in osteosarcoma patients. Expression of CBFβ contributes to the proliferation and metastatic behavior of osteosarcoma cells. Conclusions These data establish CBFβ as a mediator of CDK11p110 dependent oncogenesis and suggest that targeting the CDK11- CBFβ pathway may be a promising therapeutic strategy for osteosarcoma treatment. Graphical Abstract

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