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Notes: The authors used a yeast two-hybrid system to identify proteins that interact with the autoimmune regulator (AIRE) protein. DAXX, a multifunctional protein involved in apoptosis and transcription regulation, interacts with AIRE, as shown through coimmunoprecipitation and colocalization studies. Colocalization of AIRE and DAXX in HeLa cells was demonstrated by confocal microscopy using a Monster Green^® Fluorescent Protein-AIRE fusion protein and endogenous DAXX, which was detected using an anti-DAXX primary antibody and an anti-rabbit secondary antibody conjugated with Texas Red fluorophore. However, AIRE and DAXX did not interact in vitro in a GST pull-down assay using a GST-AIRE construct, radiolabeled DAXX protein expressed in a TNT^® system, and MagneGST™ Glutathione Particles, leading the authors to speculate that the interaction is weak or there are scaffold proteins required for protein interaction. To examine the effect of DAXX on AIRE transcriptional activity, the authors transfected COS-1 and HeLa cells with AIRE and DAXX expression constructs and a luciferase reporter plasmid with the human insulin promoter, then performed Dual Luciferase^® Reporter Assays. AIRE induced transcription of the insulin promoter, but coexpression of DAXX suppressed this transcriptional activation. (4153)

Notes: The authors developed a high-density protein array using a recombinant peptide library to map the epitope recognized by a commercially available anti-vitamin D receptor (VDR) monoclonal antibody. By screening 2304 overlapping VDR peptides, they were able to identify the 37-amino-acid epitope. The library was created by amplifying the 1.2kb VDR coding region, cleaning the PCR product with the Wizard^® SV Gel and PCR Clean-Up System, sonicating the PCR product, then cloning the VDR fragments into a bacterial expression vector that confers a glutathione-S-transferase (GST) tag. The epitope was verified by showing that the 37-amino-acid sequence was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA); the full-length VDR, also expressed as GST fusion protein, was used as a positive control. These GST fusion proteins were purified using the MagneGST™ Protein Purification System. (4154)

Notes: The authors investigated the calcium-dependent interaction between the Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) and the novel EhC2A protein. EhC2A was expressed as a GST fusion protein and immobilized on MagneGST™ Glutathione Particles for use in liposome-binding assays to show that EhC2A binds to ³H-labeled liposomes in the presence of Ca²⁺. EhC2A then was shown to interact with URE3-BP in a Ca²⁺-dependent manner to recruit URE3-BP to phospholipid liposomes. This is consistent with the observation that EhC2A and URE3-BP translocate to the amebic plasma membrane of E. histolytica trophozoites with an increase in intracellular Ca²⁺ concentration. (4151)

Notes: The authors investigated the interaction between the α1-syntrophin (SNTA1), and the α subunit (SCN5A) of the cardiac sodium channel macromolecular complex, which includes neural nitric oxide synthase (nNOS) and the plasma membrane Ca-ATPase PMCA4b. One method that the authors used to identify protein:protein interactions was glutathione-S-transferase (GST) pull-down assays. The authors purified the GST-labeled C-terminus of SCN5A using the MagneGST™ Particles and used the immobilized protein as bait to pull down the SCN5A-associated proteins from cells transfected with expression vectors SCN5A, PMCA4b or nNOS. Proteins were allowed to interact overnight at 4°C in binding buffer and washed four times before denaturation in Laemmli sample buffer and analysis by SDS-PAGE. SNTA1 interacted with the bait protein but the missense mutation (A390V-SNTA1) did not. (3897)

Notes: The authors generated a collection of >5,000 clones containing predicted protein-coding sequences from Saccharomyces cerevisiae. Each clone was sequenced to verify the open reading frame (ORF) sequence. In addition, the ORFs from 257 clones that encode known or predicted transcription factors were transferred to a bacterial expression vector and expressed as GST-fusion proteins. GST-fusion proteins were purified using MagneGST™ Glutathione Particles in a 96-well format with a Biomek^® FX automation workstation for all steps (cell lysis to elution of bound protein). Purified proteins were then applied to a protein-binding microarray to identify DNA sequences that were bound by each protein. (3783)

Notes: The authors developed a new DNA methylation detection technique based on the affinity of MBD2b and MBD3L1 proteins for CpG-methylated DNA and used this technique to examine DNA methylation of homeodomain proteins. GST-tagged MBD2b protein was incubated with genomic DNA isolated from normal or cancer cells. The GST-MBD2b protein and associated DNA were bound to MagneGST^® particles that were preblocked with JM110 bacterial DNA by incubation at 4°C for 45 minutes. Particles were washed three times in wash buffer [10mM Tris-HCl (pH7.5), 700mM NaCl, 1mM EDTA, 3mM MgCl₂, 0.1% Triton^® X-100], prior to elution of the methylated DNA. (3684)

Notes: The authors investigated the role of Protein 4.2 Komatsu mutation in hereditary spherocytosis by examining the interaction of Protein 4.2 and Protein 4.2 site-directed mutants with the membrane skeletal protein ankyrin and the cytoplasmic domain of band 3 (CDB3). Site-directed Protein 4.2 mutations were created by substituting various amino acids at residues D175 and A142, and the resulting mutant proteins were expressed in E. coli as glutathione-S-transferase fusion proteins, then purified. Interactions of these mutant proteins with ankyrin and CDB3 were examined by Far Westen blot and protein pull-down assays. Protein purification and protein pull-down assays were perfromed using the MagneGST™ Glutathione Particles. (3965)

Notes: The MagneGST™ Protein Purification System was used to purify GST fusion proteins of the oncoprotein HPV16 E7 or various mutants of HPV16 E7. The purified GST fusion proteins were used for in vitro binding experiments with steroid receptor coactivator 1 (SRC-1), which was produced using the TNT^® T7 Coupled Wheat Germ Extract System and labeled with the Transcend™ Non-Radioactive Translation Detection System. GST pull-down assays were resolved by Western analysis using streptavidn-horseradish peroxidase and alpha-GST. To determine the effects of endogenously expressed HPV16 E7 on SCR-1-mediated transcription, luciferase reporters under the control of either the IL-8 promoter or an artificial promoter containing three estrogen response elements repeats (3 × EREs) were cotransfected with a Renilla control vector into two human cervical cancer lines (C33A and CaSki) using either the Transfast™ Transfection Reagent or another commercial transfection reagent. The Dual-Luciferase^® Reporter Assay was then used to determine luciferase activity to functionally map the E7-interacting domain and to determine the effects of high- and low-risk PHV E7s on SRC-1-mediated transcription. (3459)

Notes: In this study, researchers examined the role of tumour necrosis factor (TNF) receptor-associated factors (TRAFs) in signaling by the KSHV viral FADD-like interleukin-1-β-converting enzyme (FLICE)/caspase-8-inhibitory protein (vFLIP). The TRAF domain of TRAF2 was expressed in vitro from a plasmid construct using the TNT^® Quick Coupled Transcription/Translation System and labeled with [³⁵S]methionine. vFLIP was cloned in-frame with a carboxy-terminal GST tag, and the recombinant vFLIP–GST fusion protein was expressed in E. coli and purified using the MagneGST™ Protein Purification System. GST protein only was similarly prepared as a control. The vFLIP–GST fusion and control proteins were incubated with the radiolabeled recombinant TRAF2, and protein complexes were collected by GST pull-down, washed thoroughly and subjected to SDS–polyacrylamide gel electrophoresis. In vitro-translated proteins were visualized by autoradiography. (3327)

Notes: To help determine the role of Escherichia coli glgX gene in bacterial glycogen metabolism, researchers cloned the gene into a glutathione-S-transferase expression vector, pDEST15. Expression was induced by 0.2% arabinose for 3 hours and the protein was then purified using the MagneGST™ Protein Purification System. Protein expression was monitored and analyzed by SDS-PAGE and Coomassie blue staining. (3282)