Detection of Protein‐Protein Interactions by Coprecipitation

Elion, Elaine A.

doi:10.1002/0471142727.im0807s76

Cited by 9 publications

(8 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The immunoprecipitated proteins were solubilized by boiling in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) sample buffer for 5 min and were subject to SDS‐PAGE and Western blotting. For immunoprecipitation–recapture the protocol described elsewhere (Elion, ) was followed.…”

Section: Methodsmentioning

confidence: 99%

Neuregulin‐1 promotes remyelination and fosters a pro‐regenerative inflammatory response in focal demyelinating lesions of the spinal cord

Kataria

Alizadeh

Shahriary

et al. 2017

Glia

View full text Add to dashboard Cite

Oligodendroglial cell death and demyelination are hallmarks of neurotrauma and multiple sclerosis that cause axonal damage and functional impairments. Remyelination remains a challenge as the ability of endogenous precursor cells for oligodendrocyte replacement is hindered in the unfavorable milieu of demyelinating conditions. Here, in a rat model of lysolecithin lysophosphatidyl-choline (LPC)-induced focal demyelination, we report that Neuregulin-1 (Nrg-1), an important factor for oligodendrocytes and myelination, is dysregulated in demyelinating lesions and its bio-availability can promote oligodendrogenesis and remyelination. We delivered recombinant human Nrg-1β1 (rhNrg-1β1) intraspinally in the vicinity of LPC demyelinating lesion in a sustained manner using poly lactic-co-glycolic acid microcarriers. Availability of Nrg-1 promoted generation and maturation of new oligodendrocytes, and accelerated endogenous remyelination by both oligodendrocyte and Schwann cell populations in demyelinating foci. Importantly, Nrg-1 enhanced myelin thickness in newly remyelinated spinal cord axons. Our complementary in vitro studies also provided direct evidence that Nrg-1 significantly promotes maturation of new oligodendrocytes and facilitates their transition to a myelinating phenotype. Nrg-1 therapy remarkably attenuated the upregulated expression chondroitin sulfate proteoglycans (CSPGs) specific glycosaminoglycans in the extracellular matrix of demyelinating foci and promoted interleukin-10 (IL-10) production by immune cells. CSPGs and IL-10 are known to negatively and positively regulate remyelination, respectively. We found that Nrg-1 effects are mediated through ErbB2 and ErbB4 receptor activation. Our work provides novel evidence that dysregulated levels of Nrg-1 in demyelinating lesions of the spinal cord pose a challenge to endogenous remyelination, and appear to be an underlying cause of myelin thinning in newly remyelinated axons.

show abstract

Section: Methodsmentioning

confidence: 99%

Neuregulin‐1 promotes remyelination and fosters a pro‐regenerative inflammatory response in focal demyelinating lesions of the spinal cord

Kataria

Alizadeh

Shahriary

et al. 2017

Glia

View full text Add to dashboard Cite

show abstract

“…Brush-border membrane vesicles (BBMVs) were prepared from mouse intestine as described by Biber et al (24). The immunoprecipitation (IP) procedure was adapted from Elion (25). Anti-human ACE2 antibody (ab15348) was obtained from Abcam (Cambridge, UK), which crossreacts with the mouse protein.…”

Section: Immunoprecipitationmentioning

confidence: 99%

A protein complex in the brush‐border membrane explains a Hartnup disorder allele

Kowalczuk¹,

Bröer²,

Tietze³

et al. 2008

FASEB j.

212

201

View full text Add to dashboard Cite

Protein absorption in the intestine is mediated by proteases and brush-border peptidases together with peptide and amino acid transporters. Neutral amino acids are generated by a variety of aminopeptidases and carboxypeptidases and are subsequently taken up by the amino acid transporter B(0)AT1 (SLC6A19), which is mutated in Hartnup disorder. Coexpression of B(0)AT1 together with the brush-border carboxypeptidase angiotensin-converting enzyme 2 (ACE2) in Xenopus laevis oocytes led to a dramatic increase of transporter expression at the oocyte surface. Other members of the SLC6 family were not stimulated by coexpression with ACE2. Addition of a peptide containing a carboxyterminal leucine residue to ACE2- and B(0)AT1-coexpressing oocytes caused inward currents due to Na(+)-leucine cotransport, demonstrating the formation of a metabolic complex. Coexpression of the Hartnup disorder causing mutation B(0)AT1(R240Q) showed reduced interaction with ACE2 and its renal paralogue collectrin. This would result in reduced surface expression in both kidney and intestine, thereby explaining the onset of the disorder in individuals carrying this mutation.

show abstract

“…Indeed, small variations in the relative amounts of salt and detergents in the lysis buffer as well as the speed and efficiency of cell breakage are particularly important in the isolation of proteins that associate with macromolecular structures (38). Therefore, conditions for purification of ORC(1-6) from cell lysates were reinvestigated.…”

Section: Orc(1-6) Can Be Purified From Cellmentioning

confidence: 99%

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

Ghosh

Vassilev

Zhang

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually.

show abstract

Detection of Protein‐Protein Interactions by Coprecipitation

Cited by 9 publications

References 24 publications

Neuregulin‐1 promotes remyelination and fosters a pro‐regenerative inflammatory response in focal demyelinating lesions of the spinal cord

Neuregulin‐1 promotes remyelination and fosters a pro‐regenerative inflammatory response in focal demyelinating lesions of the spinal cord

A protein complex in the brush‐border membrane explains a Hartnup disorder allele

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

Contact Info

Product

Resources

About